Nucleic acid encoding neuropeptide Y/peptide YY (Y2) receptors and uses thereof

ABSTRACT

This invention provides isolated nucleic acid molecules encoding Y2 receptors, an isolated, purified Y2 receptor protein, vectors comprising isolated nucleic acid molecules encoding Y2 receptors, mammalian, insect, bacterial and yeast cells comprising such vectors, antibodies directed to the Y2 receptors, nucleic acid probes useful for detecting nucleic acid encoding Y2 receptors, antisense oligonucleotides complementary to unique sequences of a nucleic acid molecule which encodes a Y2 receptor, pharmaceutical compounds related to the Y2 receptors, and nonhuman transgenic animals which express nucleic acid encoding a normal or mutant Y2 receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and methods of treatment involving Y2 receptors.

[0001] This application is a continuation-in-part of U.S. Ser. No.08/192,288, filed Feb. 3, 1994, the contents of which are herebyincorporated by reference into the subject application.

BACKGROUND OF THE INVENTION

[0002] Throughout this application, various publications are referencedin parenthesis by number. Full citations for these references may befound at the end of the specification immediately preceding the claims.The disclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the art to which thisinvention pertains.

[0003] Neuropeptides are small peptides originating from large precursorproteins synthesized by peptidergic neurons and endocrine/paracrinecells. They hold promise for treatment of neurological, psychiatric, andendocrine disorders (46). Often the precursors contain multiplebiologically active peptides. There is great diversity of neuropeptidesin the brain caused by alternative splicing of primary gene transcriptsand differential precursor processing. The neuropeptide receptors serveto discriminate between ligands and to activate the appropriate signals.

[0004] Neuropeptide Y (NPY), a 36-amino acid peptide, is the mostabundant neuropeptide to be identified in mammalian brain. NPY is animportant regulator in both the central and peripheral nervous systems(47) and influences a diverse range of physiological parameters,including effects on psychomotor activity, food intake, centralendocrine secretion, and vasoactivity in the cardiovascular system. Highconcentrations of NPY are found in the sympathetic nerves supplying thecoronary, cerebral, and renal vasculature and have contributed tovasoconstriction. NPY binding sites have been identified in a variety oftissues, including spleen (48), intestinal membranes, brain (49), aorticsmooth muscle (50), kidney, testis, and placenta (2). In addition,binding sites have been reported in a number of rat and human cell lines(e.g. Y1 in SK-N-MC, MC-IXC, CHP-212, and PC12 cells; Y2 in SK-N-Be(2),CHP-234, and SMS-MSN) (51,5).

[0005] Neuropeptide Y (NPY) receptor pharmacology is currently definedby structure activity relationships within the pancreatic polypeptidefamily (1, 2). This family includes NPY, which is synthesized primarilyin neurons; peptide YY (PYY), which is synthesized primarily byendocrine cells in the gut; and pancreatic polypeptide (PP), which issynthesized primarily by endocrine cells in the pancreas. These 36 aminoacid peptides have a compact helical structure involving a “PP-fold” inthe middle of the peptide. Specific features include a polyproline helixin residues 1 through 8, a β-turn in residues 9 through 14, an α-helixin residues 15 through 30, an outward-projecting C-terminus in residues30 through 36, and a carboxyl terminal amide which appears to becritical for biological activity (3). The peptides have been used todefine at least four receptor subtypes known as Y1, Y2, Y3, and PP. Y1receptor recognition by NPY involves both N- and C-terminal regions ofthe peptide; exchange of Gln³⁴ with Pro³⁴ is fairly well tolerated (3,4, 5). Y2 receptor recognition by NPY depends primarily upon the fourC-terminal residues of the peptide (Arg³-Gln³⁴-Arg³⁵-Tyr³⁶-NH₂) precededby an amphipathic α-helix (3, 6, 7); exchange of Gln₃₄ with Pro³⁴ is notwell tolerated (4, 5). Y3 receptor recognition is characterized by astrong preference for NPY over PYY (8). Exchange of Gln₃₄ in NPY withPro³⁴ is reasonably well tolerated by the Y3 receptor but PP, which alsocontains Pros, does not bind well (8). The PP receptor is reported tobind tightly to PP, less so to [Leu³¹,Pro³⁴]NPY, and even less so to NPY(3, 9). The only NPY receptor which has been cloned to date is the Y1receptor gene, from mouse (12), rat (52), and human (10). One of the keypharmacological features which distinguish Y1 and Y2 is the fact thatthe Y1 receptor (and not the Y2 receptor) responds to an analog of NPYmodified at residues 31 and 34 ([Leu31,Pro34]NPY), whereas the Y2receptor (and not the Y1 receptor) has high affinity for the NPY peptidecarboxyl-terminal fragment NPY-(13-36) (53,4).

[0006] Receptor genes for the other two structurally related peptides,peptide YY (PYY) and pancreatic polypeptide (PP), also have not beencloned. Peptide YY occurs mainly in endocrine cells in the lowergastrointestinal tract (54). Receptors for PYY were first described inthe rat small intestine (55). This receptor has been defined asPYY-preferring because it displays a 5-10 fold higher affinity for PYYthan for NPY (55,56). Recently, a cell line, PKSV-PCT, derived from theproximal tubules of kidneys, has been described to express receptors forPYY (57).

[0007] In the last few years only the rat and human Y1 cDNAs have beencloned (10, 11). This success was based on identifying the randomlycloned FC5 “orphan receptor” (12). We now report the isolation byexpression cloning of a human hippocampal Y2 cDNA clone and two rat Y2clones and their pharmacological characterization.

SUMMARY OF THE INVENTION

[0008] This invention provides an isolated nucleic acid moleculeencoding a Y2 receptor.

[0009] This invention also provides an isolated protein which is a Y2receptor.

[0010] This invention provides a vector comprising nucleic acid encodinga Y2 receptor. This invention also provides vectors such as plasmidscomprising nucleic acid encoding a Y2 receptor, adapted for expressionin a bacterial cell, a yeast cell, an insect cell or a mammalian cellwhich additionally comprise the regulatory elements necessary forexpression of the nucleic acid in the bacterial, yeast, insect ormammalian cells operatively linked to the nucleic acid encoding the Y2receptor as to permit expression thereof.

[0011] This invention provides a cell transfected with and expressingnucleic acid encoding a Y2 receptor.

[0012] This invention provides a nucleic acid probe comprising a nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with a unique sequence included within the sequence of anucleic acid molecule encoding a Y2 receptor.

[0013] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing with any sequences of anmRNA molecule which encodes a Y2 receptor so as to prevent translationof the mRNA molecule.

[0014] This invention provides an antibody directed to a Y2 receptor.

[0015] This invention provides a transgenic nonhuman mammal expressingnucleic acid encoding a Y2 receptor. This invention further provides atransgenic nonhuman mammal whose genome comprises antisense DNAcomplementary to DNA encoding a Y2 receptor so placed as to betranscribed into antisense mRNA which is complementary to mRNA encodinga Y2 receptor and which hybridizes to mRNA encoding a Y2 receptorthereby reducing its translation.

[0016] This invention further provides a transgenic nonhuman mammalcomprising a homologous recombination knockout of the native Y2receptor.

[0017] This invention provides a method for determining whether a ligandcan bind specifically to a Y2 receptor which comprises contacting a celltransfected with and expressing nucleic acid encoding the Y2 receptorwith the ligand under conditions permitting binding of ligands to suchreceptor, and detecting the presence of any such ligand bound to the Y2receptor, thereby determining whether the ligand binds specifically to aY2 receptor.

[0018] This invention also provides a method for determining whether aligand is a Y2 receptor agonist which comprises contacting a celltransfected with and expressing nucleic acid encoding the Y2 receptorwith the ligand under conditions permitting the activation of afunctional Y2 receptor response from the cell, and detecting, by meansof a bioassay, such as a second messenger assay, an increase in Y2receptor activity, thereby determining whether the ligand is a Y2receptor agonist.

[0019] This invention further provides a method for determining whethera ligand is a Y2 receptor antagonist which comprises contacting a celltransfected with and expressing nucleic acid encoding the Y2 receptorwith the ligand in the presence of a known Y2 receptor agonist, such asNPY, under conditions permitting the activation of a functional Y2receptor response, and detecting, by means of a bioassay, such as asecond messenger assay, a decrease in Y2 receptor activity, therebydetermining whether the ligand is a Y2 receptor antagonist.

[0020] This invention further provides a method of screening drugs toidentify drugs which specifically bind to a Y2 receptor on the surfaceof a cell which comprises contacting a cell transfected with andexpressing nucleic acid encoding the Y2 receptor with a plurality ofdrugs under conditions permitting binding of drugs to the Y2 receptor,and determining those drugs which bind to the Y2 receptor, therebyidentifying drugs which specifically bind to a Y2 receptor.

[0021] This invention also provides a method of screening drugs toidentify drugs which act as agonists of a Y2 receptor on the surface ofa cell which comprises contacting a cell transfected with and expressingnucleic acid encoding the Y2 receptor with a plurality of drugs underconditions permitting the activation of a functional Y2 receptorresponse, and determining those drugs which activate the Y2 receptor,using a bioassay, such as a second messenger assay, thereby identifyingdrugs which act as Y2 receptor agonists.

[0022] This invention also provides a method of screening drugs toidentify drugs which act as antagonists of a Y2 receptor on the surfaceof a cell which comprises contacting a cell transfected with andexpressing nucleic acid encoding the Y2 receptor with a plurality ofdrugs in the presence of a known Y2 receptor agonist, such as NPY, underconditions permitting the activation of a functional Y2 receptorresponse, and determining those drugs which inhibit the activation ofthe Y2 receptor, using a bioassay, such as a second messenger assay,thereby identifying drugs which act as Y2 receptor antagonists.

[0023] This invention also provides a method of detecting expression ofa Y2 receptor by a cell by detecting the presence of mRNA coding for theY2 receptor which comprises obtaining total mRNA from the cell andcontacting the mRNA so obtained with a nucleic acid probe comprising anucleic acid molecule of at least 15 nucleotides capable of specificallyhybridizing with a unique sequence included within the sequence of anucleic acid molecule encoding the Y2 receptor under hybridizingconditions, and detecting the presence of mRNA hybridized to the probe,thereby detecting the expression of a Y2 receptor by the cell.

[0024] This invention provides a method of determining the physiologicaleffects of expressing varying levels of Y2 receptors which comprisesproducing a transgenic nonhuman mammal expressing nucleic acid encodinga Y2 receptor whose levels of Y2 receptor expression are varied by useof an inducible promoter which regulates Y2 receptor expression.

[0025] This invention also provides a method of determining thephysiological effects of expressing varying levels of Y2 receptors whichcomprises producing a panel of transgenic nonhuman animals eachexpressing nucleic acid encoding a Y2 receptor expressing nucleic acidand expressing a different amount of Y2 receptor.

[0026] This invention provides a method for diagnosing a predispositionto a disorder associated with the activity of a specific Y2 receptorallele which comprises: a. obtaining nucleic acid of subjects sufferingfrom the disorder; b. performing a restriction digest of the nucleicacid with a panel of restriction enzymes; c. electrophoreticallyseparating the resulting nucleic acid fragments on a sizing gel; d.contacting the resulting gel with a nucleic acid probe capable ofspecifically hybridizing to nucleic acid encoding a Y2 receptor andlabeled with a detectable marker; e. detecting labeled bands which havehybridized to the nucleic acid encoding a Y2 receptor labelled with adetectable marker to create a unique band pattern specific to thenucleic acid of subjects suffering from the disorder; f. preparingnucleic acid obtained for diagnosis by steps a-e; and g. comparing theunique band pattern specific to the nucleic acid of subjects sufferingfrom the disorder from step e and the nucleic acid obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.

[0027] This invention provides a method of preparing an isolated,purified Y2 receptor which comprises constructing a vector adapted forexpression in a cell which comprises the regulatory elements necessaryfor the expression of nucleic acid in the cell operatively linked to thenucleic acid encoding a Y2 receptor as to permit expression thereof,wherein the cell is selected from the group consisting of bacterialcells, yeast cells, insect cells and mammalian cells; inserting thevector of the previous step in a suitable host cell; incubating thecells under conditions allowing the expression of a Y2 receptor;recovering the receptor so produced and purifying the receptor sorecovered.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1

[0029] Nucleotide sequence of the human hippocampal Y2 cDNA clone (SEQ.I.D. No. 1). Initiation and stop codon are indicated in bold. Onlypartial 5′ and 3′ untranslated sequences are shown.

[0030]FIG. 2

[0031] Deduced amino acid sequence of the human hippocampal Y2 cDNAclone encoded by the nucleotide sequence in FIG. 1 (SEQ. I.D. No. 2).

[0032] FIGS. 3-1 through FIGS. 3-4

[0033] Comparison of coding nucleotide sequences between humanhippocampal Y2 (top row) and Y1 human cDNA clones (bottom row) (48.5%nucleotide identity).

[0034] FIGS. 4-1 and FIGS. 4-2

[0035] Comparison of amino acid sequences between hippocampal Y2 (toprow) and Y1 human cDNA clones (bottom row). (31% overall identity and41% in the transmembrane domains).

[0036]FIG. 5A

[0037] Equilibrium binding of ¹²⁵I-PYY to membranes from COS-7 cellstransiently expressing CG-13 () and human Y1 (∘) receptors. Membraneswere incubated with ¹²⁵I-PYY for the times indicated, in the presence orabsence of 100 nM human NPY. Specific binding, B, was plotted againsttime, t, to obtain the maximum number of equilibrium binding sites, B₁and B₂, and observed association rates, K_(obs1) and K_(obs2), accordingto the equation, B=B₁*(1−e^(−(kobs1*t)))+B₂*(1−e^(−(kobs2*t))). Bindingis shown as the percentage of total equilibrium binding, B₁+B₂,determined by nonlinear regression analysis. Data are representative ofthree independent experiments, with each point measured in triplicate.

[0038]FIG. 5B

[0039] Equilibrium binding of ¹²⁵I-PYY to membranes from COS-7 cellstransiently expressing CG-13 () and human Y1 (∘) receptors using thesame conditions as in FIG. 5A except for a prolonged time course of upto 180 minutes.

[0040]FIG. 6

[0041] Saturable equilibrium binding of ¹²⁵I-PYY to membranes from COS-7cells transiently expressing CG-13 receptors. Membranes were incubatedwith ¹²⁵I-PYY ranging in concentration from 0.003 nM to 2 nM, in thepresence or absence of 100 nM human NPY. Specific binding, B, wasplotted against the free ¹²⁵I-PYY concentration, [L], to obtain themaximum number of saturable binding sites, B_(max), and the ¹²⁵I-PYYequilibrium dissociation constant, K_(d), according to the bindingisotherm, B=B_(max)[L]/([L]+K_(d)). Specific binding is shown. Data arerepresentative of three independent experiments, with each pointmeasured in triplicate.

[0042]FIG. 7A

[0043] Competitive displacement of ¹²⁵I-PYY on membranes from COS-7cells transiently expressing Human Y1 receptors. Membranes wereincubated with ¹²⁵I-PYY and increasing concentrations of peptidecompetitors. IC₅₀ values corresponding to 50% displacement weredetermined by nonlinear regression analysis and converted to K_(i)values according to the equation, K_(i)=IC₅₀(1+[L]/K_(d)), where [L] isthe ¹²⁵I-PYY concentration and K_(d) is the equilibrium dissociationconstant of ¹²⁵I-PYY. Data are representative of at least twoindependent experiments, with each point measured once or in duplicate.Rank orders of affinity for these and other compounds are listedseparately in Table 2.

[0044]FIG. 7B

[0045] Competitive displacement of ¹²⁵I-PYY on membranes from COS-7cells transiently expressing human Y2 receptors. Membranes wereincubated with ¹²⁵I-PYY and increasing concentrations of peptidecompetitors. IC₅₀ values corresponding to 50% displacement weredetermined by nonlinear regression analysis and converted to K_(i)values according to the equation, K_(i)=IC₅₀(1+[L]/K_(d)), where [L] isthe ¹²⁵I-PYY concentration and K_(d) is the equilibrium dissociationconstant of ¹²⁵I-PYY. Data are representative of at least twoindependent experiments, with each point measured once or in duplicate.Rank orders of affinity for these and other compounds are listedseparately in Table 2.

[0046]FIG. 8 Nucleotide sequence (SEQ. I.D. No. 3) and deduced aminoacid sequence (SEQ. I.D. No. 4) of the rat Y2 receptor encoded by rs5a.Nucleotides are presented in the 5′ to 3′ orientation and the codingregion is numbered starting from the putative initiating methionine andending in the termination codon. Deduced amino acid sequence bytranslation of a long open reading frame is shown using one-lettersymbols.

[0047]FIG. 9 Nucleotide sequence (SEQ. I.D. No. 5) and deduced aminoacid sequence (SEQ. I.D. No. 6) of the rat Y2 receptor encoded by rs26a.Nucleotides are presented in the 5′ to 3′ orientation and the codingregion is numbered starting from the putative initiating methionine andending in the termination codon. Deduced amino acid sequence bytranslation of a long open reading frame is shown using one-lettersymbols.

[0048]FIG. 10 Alignment of rat and human Y2 receptors: nucleotidesequences. Nucleotide sequences of the ceding regions of the human Y2receptor (HumY2) and the rat Y2 receptors encoded by rs5a (RatY2a) andrs26a (RatY2b) are shown; the nucleotide sequence of rs26a (RatY2b) isidentical to rs5a (RatY2a) except where shown. Rat and human Y2nucleotide sequences exhibit ˜86% identity in the coding region.

[0049]FIG. 11 Alignment of rat and human Y2 receptors: amino acidsequences. Complete predicted amino acid sequences of the human Y2receptor (Hum Y2) and the rat Y2 receptor encoded by rs5a (Rat Y2a) areshown; the amino acid sequence of RatY2b encoded by rs26a is identicalto RatY2a except where shown. Rat and human Y2 amino acid sequences are˜94% identical overall and ˜98% identical in the transmembrane domains(bracketed). Single letter abbreviations for amino acids are shown.

[0050]FIG. 12 Localization of Rat Y2 mRNA in the rat central nervoussystem. Schematic diagrams of half-coronal sections through the ratbrain showing the distribution of neuropeptide Y Y2 receptor mRNAobtained with radiolabelled oligonucleotide probes and in situhybridization histochemistry. The stars show the location of labeledneuronal populations, and are not indicative of the number of cellsobserved in each area.

[0051]FIG. 13 Effects of Gpp(NH)p on radio ligand binding to Y2receptors. Binding data were generated from competitive displacementassays in the absence () or presence (∘) of 100 μM Gpp(NH)p. Themaximum specific binding detected under control conditions (in theabsence of Gpp(NH)p) was used to normalize the data. A) Human Y2receptor transiently expressed in COS-7 cells. B) Rat Y2a receptortransiently expressed in COS-7 cells.

[0052]FIG. 14 Inhibition of forskolin-stimulated cAMP accumulation inintact cells stably expressing the human Y2 receptor. Functional datawere derived from radioimmunoassay of CAMP in 293 cells stimulated with10 μM forskolin over a 5 min period. Human PYY was tested for agonistactivity over the same period. Data were fit to a four parameterlogistic equation by nonlinear regression. Data generated from stablytransfected 293 cells () and from stably transfected NIH-3T3 cells (∘).Data shown are representative of ten () and two (∘) independentexperiments.

[0053]FIG. 15 Stimulation of intracellular free calcium concentration inintact 293 cells stably expressing the human Y2 receptor. A) Timecourse. Functional data were derived from Fura-2 μM fluorescence in 293cells stimulated with 1 μM human PYY at the time indicated by the arrow.B) Time course. Cells were stimulated with 1 μM human PYY as in A exceptthat 1 mM EGTA was included in the extracellular solution. C)Concentration/response curve for PYY-dependent mobilization ofintracellular calcium in 293 cells stably transfected with the human Y2receptor. Data were fit to a four parameter logistic equation bynonlinear regression. Data shown are representative of at least twoindependent experiments.

[0054]FIG. 16. Northern analysis of various human brain areas.Hybridization was done under conditions of high stringency, as describedin Experimental Details. The probe was a ³²P-labeled DNA fragment(specific activity 3×10⁹ cpm/μg) corresponding to the entire codingregion (as shown in FIG. 10) of the human NPY Y2 recepotr. The BRL RNAladder was used as molecular weight markers.

[0055]FIG. 17. Southern analysis of genomic DNA encoding the human NPYY2 receptor subtype. Hybridization was done under conditions of highstringency, as described in Experimental Details. The probe was a³²P-labeled DNA fragment (specific activity 2.5×10⁹ cpm/μg)corresponding to the TM1-TM5 region of the human NPY Y2 receptor (asshown in FIG. 11). Hind III digested λ DNA was used as molecular weightmarkers.

[0056]FIG. 18. Photomicrographs showing some of the controls used forNPY Y2 oligonucleotide probe specificity (A, B), and tissue distributionof the hybridization signal in rat brain (C, D). A. Darkfieldphotomicrograph of the hybridization signal obtained using theradiolabeled antisense probe on COS-7 cells transfected with the rat Y2DNA. B. Hybridization signal obtained following hybridization with theradiolabeled sense probe, also on transfected COS-7 cells. Only theantisense probes hybridize to the transfected cells. C. Brightfieldphotomicrograph of the hybridization signal observed in the CA3 regionof the rat hippocampus. Silver grains are found over neuronal cellbodies (arrows) in the pyramidal cell layer (sp), but not over thestratum lucidum (slu) or stratum radiatum (sr). D. Hybridization signalobserved over neurons (arrows) in the arcuate nucleus of thehypothalamus. The darkly stained ependymal lining of the third ventriclecan be seen to the left of the micrograph (asterisk).

DETAILED DESCRIPTION OF THE INVENTION

[0057] Throughout this application, the following standard abbreviationsare used to indicate specific nucleotide bases: C = cytosine A = adenineT = thymine G = guanine

[0058] This invention provides isolated nucleic acid molecules whichencode Y2 receptors. In one embodiment, the Y2 receptor encoded is ahuman Y2 receptor. In another embodiment, the Y2 receptor encoded is arat Y2 receptor. As used herein, the term Y2 receptor encompasses anyamino acid sequence, polypeptide or protein having substantially thesame pharmacology provided for the subject human Y2 receptor as shown inTables 2-4 and FIGS. 5A-7B. As described herein our cloned receptor hasa Y2 pharmacological profile that differs from the NPY receptor subtypesY1 and Y3, PYY receptor, and PP receptor, and is therefore designated asthe Y2 receptor.

[0059] The only NPY receptor which has been cloned to date is the Y1receptor gene, from mouse (Eva et al., 1992), rat (Eva et al., 1990),and human (Larhammar et al., 1992). The human Y2 receptor's greatesthomology with any known receptor disclosed in the Genbank/EMBL®databases is a 42% overall amino acid identity with the human Y1receptor.

[0060] This invention provides isolated nucleic acid molecules encodingY2 receptors. In one embodiment, the Y2 receptor is a human Y2 receptor.In another embodiment, the Y2 receptor is a rat Y2 receptor. As usedherein, the term “isolated nucleic acid molecule” means a nucleic acidmolecule that is a molecule in a form which does not occur in nature.Examples of such an isolated nucleic acid molecule are an RNA, cDNA, orisolated genomic DNA molecule encoding a Y2 receptor. The human Y2receptor has an amino acid sequence substantially the same as thededuced amino acid sequence shown in FIG. 2 and any human receptorhaving substantially the same amino acid sequence as the amino acidsequence shown in FIG. 2 is by definition a human Y2 receptor. The ratY2 receptor has an amino acid sequence substantially the same as thededuced amino acid sequences shown in FIG. 8 or FIG. 9. One means ofisolating another Y2 receptor is to probe a genomic library with anatural or artificially designed DNA probe, using methods well known inthe art. DNA probes derived from the human and the rat receptor Y2 geneare particularly useful probes for this purpose. DNA and cDNA moleculeswhich encode Y2 receptors may be used to obtain genomic DNA, cDNA or RNAfrom human, mammalian or other animal sources, or to isolate relatedcDNA or genomic clones by the screening of cDNA or genomic libraries bymethods described in more detail below. Transcriptional regulatoryelements from the 5′ untranslated region of the isolated clones, andother stability, processing, transcription, translation, and tissuespecificity-determining regions from the 3′ and 5′ untranslated regionsof the isolated genes are thereby obtained. Examples of a nucleic acidmolecule are an RNA, cDNA, or isolated genomic DNA molecule encoding aY2 receptor. Such molecules may have coding sequences substantially thesame as the coding sequences shown in FIGS. 1, 8 and 9. The DNA moleculeof FIG. 1 encodes the sequence of the human Y2 receptor gene. The DNAmolecules of FIGS. 8 and 9 encode the sequence of two rat Y2 receptorgenes.

[0061] This invention further provides DNA molecules encoding Y2receptors having coding sequences substantially the same as the codingsequences shown in FIGS. 1, 8 and 9. These molecules are obtained by themeans described above.

[0062] This invention also provides an isolated nucleic acid moleculeencoding a Y2 receptor wherein the nucleic acid molecule encodes areceptor being characterized by an amino acid sequence in thetransmembrane region, which amino acid sequence has 60% homology orhigher to the amino acid sequence in the transmembrane region of thehuman Y2 receptor as shown in FIG. 11.

[0063] This invention also provides purified isolated proteins which areY2 receptors. In one embodiment, the Y2 receptor protein is a human Y2receptor protein. In another embodiment, the Y2 receptor protein is arat Y2 receptor protein. As used herein, the term “isolated protein”means a protein molecule free of other cellular components. Examples ofsuch proteins are isolated proteins having substantially the same aminoacid sequence as the amino acid sequences shown in FIGS. 2, 8, and 9,which are a human Y2 receptor and two rat Y2 receptors, respectively.One means for obtaining an isolated Y2 receptor is to express DNAencoding the receptor in a suitable host, such as a bacterial, yeast,insect or mammalian cell, using methods well known in the art, andrecovering the receptor protein after it has been expressed in such ahost, again using methods well known in the art. The receptor may alsobe isolated from cells which express it, in particular from cells whichhave been transfected with the expression vectors described below inmore detail.

[0064] This invention provides vectors comprising nucleic acid moleculessuch as DNA, RNA, or cDNA encoding Y2 receptors. In one embodiment, thenucleic acid encodes a human Y2 receptor. In another embodiment, thenucleic acid encodes a rat Y2 receptor. Examples of vectors are virusessuch as bacteriophages (such as phage lambda), animal viruses (such asHerpes virus, Murine Leukemia virus, and Baculovirus), cosmids, plasmids(such as pUC18, available from Pharmacia, Piscataway, N.J.), and otherrecombination vectors. Nucleic acid molecules are inserted into vectorgenomes by methods well known in the art. For example, insert and vectorDNA can both be exposed to a restriction enzyme to create complementaryends on both molecules which base pair with each other and are thenligated together with a ligase. Alternatively, linkers can be ligated tothe insert DNA which correspond to a restriction site in the vector DNA,which is then digested with the restriction enzyme which cuts at thatsite. Other means are also available. Specific examples of such plasmidsare: a plasmid comprising cDNA having a coding sequence substantiallythe same as the coding sequence shown in FIG. 1 and designated cloneCG-13 (Seq. I.D. No. 1); or a plasmid comprising genomic DNA having acoding sequence substantially the same as the coding sequence shown inFIG. 8 and designated clone rS5a (Seq. I.D. No. 3), or the codingsequence shown in FIG. 9 and designated clone rS26a (Seq. I.D. No. 5).

[0065] This invention also provides vectors comprising nucleic acidmolecules encoding Y2 receptors, adapted for expression in a bacterialcell, a yeast cell, an insect cell or a mammalian cell whichadditionally comprise the regulatory elements necessary for expressionof the nucleic acid in the bacterial, yeast, insect or mammalian cellsoperatively linked to the nucleic acid encoding a Y2 receptor as topermit expression thereof. Nucleic acid having coding sequencessubstantially the same as the coding sequence shown in FIG. 1 may beusefully inserted into the vectors to express human Y2 receptors.Nucleic acid having coding sequences substantially the same as thecoding sequences shown in FIGS. 8 and 9 may be usefully inserted intovectors to express rat Y2 receptors. Regulatory elements required forexpression include promoter sequences to bind RNA polymerase andtranscription initiation sequences for ribosome binding. For example, abacterial expression vector includes a promoter such as the lac promoterand for transcription initiation the Shine-Dalgarno sequence and thestart codon AUG (Maniatis, et al., Molecular Cloning, Cold Spring HarborLaboratory, 1982). Similarly, a eukaryotic expression vector includes aheterologous or homologous promoter for RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Furthermore, an insect expression vector,such as recombinant baculovirus, uses the polyhedron gene expressionsignals for expression of the inserted gene in insect cells. Suchvectors may be obtained commercially or assembled from the sequencesdescribed by methods well known in the art, for example the methodsdescribed above for constructing vectors in general. Expression vectorsare useful to produce cells that express the receptor. Certain uses forsuch cells are described in more detail below.

[0066] This invention further provides a plasmid adapted for expressionin a bacterial cell, a yeast cell, an insect cell, or, in particular, amammalian cell which comprises a nucleic acid molecule encoding a Y2receptor and the regulatory elements necessary for expression of thenucleic acid in the bacterial, yeast, insect, or mammalian celloperatively linked to the nucleic acid encoding the Y2 receptor as topermit expression thereof. In one embodiment, the Y2 receptor is a humanY2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor. Some plasmids adapted for expression in a mammalian cell arepSVL (available from Pharmacia, Piscataway, N.J.) and pcEXV-3 (73). Onespecific example of such a plasmid is a plasmid adapted for expressionin a mammalian cell comprising cDNA having a coding sequencesubstantially the same as the coding sequence shown in FIG. 1 and theregulatory elements necessary for expression of the DNA in the mammaliancell which is designated pcEXV-hY2, deposited on Jan. 27, 1994 underATCC Accession No. 75659. Other specific examples of such plasmids areplasmids adapted for expression in a mammalian cell comprising genomicDNA having coding sequences substantially the same as the codingsequences shown in FIGS. 8 and 9 and the regulatory elements necessaryfor expression of the DNA in the mammalian cell which are designatedpcEXV-rY2a, deposited on Jan. 25, 1995 under ATCC Accession No. 97035;and pcEXV-rY2b, deposited on Jan. 25, 1995 under ATCC Accession No.97036, respectively. Those skilled in the art will readily appreciatethat numerous plasmids adapted for expression in a mammalian cell whichcomprise DNA encoding Y2 receptors and the regulatory elements necessaryto express such DNA in the mammalian cell may be constructed utilizingexisting plasmids and adapted as appropriate to contain the regulatoryelements necessary to express the DNA in the mammalian cell. Theplasmids may be constructed by the methods described above forexpression vectors and vectors in general, and by other methods wellknown in the art.

[0067] The deposits discussed supra, and the other deposits discussedherein, were made pursuant to, and in satisfaction of, the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.

[0068] This invention provides a cell transfected with and expressingnucleic acid encoding a Y2 receptor. In one embodiment the Y2 receptoris a human Y2 receptor. In another embodiment, the Y2 receptor is a ratY2 receptor. An example of such a cell is a mammalian cell transfectedwith a plasmid adapted for expression in a mammalian cell, whichcomprises nucleic acid encoding a Y2 receptor, and the regulatoryelements necessary for expression of the nucleic acid in the mammaliancell operatively linked to the nucleic acid encoding a Y2 receptor as topermit expression thereof; the protein encoded thereby expressed on thecell surface. Numerous mammalian cells may be used as hosts, including,for example, the mouse fibroblast cell NIH-3T3, CHO cells, HeLa cells,LM (tk-) cells, etc. Expression plasmids such as that described supramay be used to transfect cells by methods well known in the art such ascalcium phosphate precipitation, or DNA encoding these Y2 receptors maybe otherwise introduced into cells, e.g., by microinjection, to obtainmammalian cells which comprise nucleic acid, e.g., cDNA or a plasmid,encoding a Y2 receptor. A specific example of such cells is a cellcomprising the pcEXV-hY2 plasmid adapted for expression in a mammaliancell comprising cDNA encoding the Y2 receptor and the regulatoryelements necessary for expression of the DNA in the mammalian cell,which is designated 293-hY2-10 and deposited on Jan. 27, 1994 under ATCCAccession No. 11837. Another specific example of such cells is a cellcomprising the pcEXV-hY2 plasmid adapted for expression in a mammaliancell comprising cDNA encoding the Y2 receptor and the regulatoryelements necessary for expression of the DNA in the mammalian cell,which is designated N-hY2-5 and deposited on Jan. 25, 1995 under ATCCAccession No. CRL-11825.

[0069] This invention provides a method for determining whether a ligandcan bind specifically to a Y2 receptor which comprises contacting a celltransfected with and expressing nucleic acid encoding a Y2 receptor, theprotein encoded thereby is expressed on the cell surface, with theligand under conditions permitting binding of ligands known to bind tothe Y2 receptor, and detecting the presence of any of the ligand boundto the Y2 receptor, thereby determining whether the ligand bindsspecifically to the Y2 receptor. In one embodiment, the Y2 receptor is ahuman Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

[0070] This invention further provides a method for determining whethera ligand can bind specifically to a Y2 receptor, which comprisescontacting a cell transfected with and expressing nucleic acid encodingthe Y2 receptor with the ligand under conditions permitting binding ofligands to such receptor, and detecting the presence of any such ligandbound to the Y2 receptor, wherein the Y2 receptor is characterized by anamino acid sequence in the transmembrane region, such amino acidsequence having 60% homology or higher to the amino acid sequence in thetransmembrane region of the Y2 receptor shown in FIG. 11. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor.

[0071] This invention provides a method for determining whether a ligandcan bind specifically to a Y2 receptor which comprises preparing a cellextract from cells transfected with and expressing nucleic acid encodinga Y2 receptor, isolating a membrane fraction from the cell extract,contacting the ligand with the membrane fraction from the cell extractunder conditions permitting binding of ligands to such receptor, anddetecting the presence of any ligand bound to the Y2 receptor, therebydetermining whether the compound is capable of binding specifically to aY2 receptor. In one embodiment, the Y2 receptor is a human Y2 receptor.In another embodiment, the Y2 receptor is a rat Y2 receptor.

[0072] This invention also provides a method for determining whether aligand is a Y2 receptor agonist. As used herein, the term “agonist”means any ligand capable of increasing Y2 receptor functional activity.This comprises contacting a cell transfected with and expressing nucleicacid encoding a Y2 receptor with the ligand under conditions permittingthe activation of a functional Y2 receptor response from the cell, anddetecting, by means of a bioassay, such as a second messenger assay, anincrease in Y2 receptor activity, thereby determining whether the ligandacts as a Y2 receptor agonist. In one embodiment, the Y2 receptor is ahuman Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

[0073] This invention further provides a method for determining whethera ligand is a Y2 receptor agonist which comprises preparing a cellextract from cells transfected with and expressing nucleic acid encodinga Y2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction of the extract with the ligand underconditions permitting the activation of a functional Y2 receptorresponse, and detecting, by means of a bioassay, such as a secondmessenger assay, an increase in Y2 receptor activity, therebydetermining whether the ligand is a Y2 receptor agonist. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor.

[0074] This invention also provides a method for determining whether aligand a Y2 receptor antagonist. As used herein, the term “antagonist”means any ligand capable of decreasing Y2 receptor functional activity.This comprises contacting a cell transfected with and expressing nucleicacid encoding a Y2 receptor with the ligand in the presence of a knownY2 receptor agonist such as NPY, under conditions permitting theactivation of a functional Y2 receptor response, and detecting, by meansof a bioassay, such as a second messenger assay, a decrease in Y2receptor activity, thereby determining whether the ligand is a Y2receptor antagonist. In one embodiment, the Y2 receptor is a human Y2receptor. In another embodiment, the Y2 receptor is a rat Y2 receptor.

[0075] This invention also provides a method for determining whether aligand is a Y2 receptor antagonist which comprises preparing a cellextract from cells transfected with and expressing nucleic acid encodinga Y2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction of the extract with the ligand in thepresence of a known Y2 receptor agonist, such as NPY, under conditionspermitting the activation of a functional Y2 receptor response, anddetecting, by means of a bioassay, such as a second messenger assay, adecrease in Y2 receptor activity, thereby determining whether the ligandis a Y2 receptor antagonist. In one embodiment, the Y2 receptor is ahuman Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

[0076] In one embodiment, the second messenger assays referred tocomprise measurement of intracellular cAMP. In another embodiment, thesecond messenger assays comprise measurement of intracellular calciummobilization.

[0077] In one embodiment, the nucleic acid in the cells referred toabove encodes a Y2 receptor having an amino acid sequence substantiallythe same as the amino acid sequence shown in FIG. 2. In anotherembodiment, the nucleic acid in the cells referred to above encodes a Y2receptor having an amino acid sequence substantially the same as theamino acid sequences shown in FIG. 8 or FIG. 9. In one embodiment, thecell is a mammalian cell. Preferably, the mammalian cell is non-neuronalin origin. An example of a nonneuronal mammalian cell is a COS-7 cell.Other examples of a non-neuronal mammalian cells that can be used forfunctional assays with Y2 receptors are the 293 human embryonic kidneycells, mouse embryonic fibroblast NIH-3T3 cells, and LM(tk-) cells.

[0078] The preferred method for determining whether a ligand is capableof binding specifically to a Y2 receptor comprises contacting atransfected nonneuronal mammalian cell (i.e. a cell that does notnaturally express any type of NPY, PP, or PYY receptor, and thus willonly express such a receptor if it is transfected into the cell)expressing a Y2 receptor on its surface, or contacting a membranepreparation derived from such a transfected cell, with the ligand underconditions which are known to prevail, and thus to be associated with,in vivo binding of the ligand to and/or activation of a Y2 receptor, anddetecting the presence of any of the ligand being tested bound to the Y2receptor on the surface of the cell, or detecting activation of the Y2receptor, thereby determining whether the ligand binds to, activates orinhibits the activation of the Y2 receptor. Activation of a Y2 receptormay be detected by means of a second messenger assay. Such a responsesystem is obtained by transfection of nucleic acid into a suitable hostcell containing the desired second messenger system such asphospholipase C, adenylate cyclase, guanylate cyclase or ion channels. Asuitable host cell can be isolated from pre-existing cell lines, or canbe generated by inserting appropriate components of second messengersystems into existing cell lines. Such a transfected cell provides acomplete response system for investigation or assay of the activity ofY2 receptors with ligands as described above. Transfection systems areuseful as living cell cultures for competitive binding assays betweenknown or candidate drugs and ligands which bind to the receptor andwhich are labeled by radioactive, spectroscopic or other reagents.Membrane preparations containing the receptor isolated from transfectedcells are also useful for Y2 receptor activity and competitive bindingassays. Functional assays of signal transduction pathways intransfection systems determine a ligand's efficacy of activating thereceptor. A transfection system constitutes a “drug discovery system”useful for the identification of natural or synthetic compounds withpotential for drug development that can be further modified or useddirectly as therapeutic compounds to activate or inhibit the naturalfunctions of the Y2 receptor. The transfection system is also useful fordetermining the affinity and efficacy of known drugs at the Y2 receptorsites.

[0079] This invention provides a pharmaceutical composition comprisingan effective amount of the Y2 receptor agonist determined by the methodsdescribed above and a pharmaceutically acceptable carrier. As usedherein, the term “pharmaceutically acceptable carrier” encompasses anyof the standard pharmaceutical carriers, such as a phosphate bufferedsaline solution, water, and emulsions, such as an oil/water or water/oilemulsion, and various types of wetting agents. In one embodiment, the Y2receptor is a human Y2 receptor. In another embodiment, the Y2 receptoris a rat Y2 receptor. In a further embodiment, the Y2 receptor agonistis not previously known.

[0080] This invention further provides a pharmaceutical compositioncomprising an effective amount of the Y2 receptor antagonist determinedby the methods described above and a pharmaceutically acceptablecarrier. In one embodiment the Y2 receptor is a human Y2 receptor. Inanother embodiment, the Y2 receptor is a rat Y2 receptor. In a furtherembodiment, the Y2 receptor antagonist is not previously known.

[0081] This invention also provides a method of screening drugs toidentify drugs which specifically bind to a Y2 receptor on the surfaceof a cell which comprises contacting a cell transfected with andexpressing nucleic acid encoding the Y2 receptor with a plurality ofdrugs under conditions permitting binding of drugs to the Y2 receptor,and determining those drugs which bind specifically to the cell, therebyidentifying drugs which specifically bind to a Y2 receptor. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor.

[0082] This invention also provides a method of screening drugs toidentify drugs which specifically bind to a Y2 receptor on the surfaceof a cell which comprises preparing a cell extract from the cellstransfected with and expressing nucleic acid encoding the Y2 receptor,isolating a membrane fraction from the cell extract, contacting themembrane fraction with a plurality of drugs under conditions permittingbinding of drugs to the Y2 receptor, and determining those drugs whichbind specifically to the transfected cell, thereby identifying drugswhich bind specifically to a Y2 receptor. In one embodiment, the Y2receptor is a human Y2 receptor. In another embodiment, the Y2 receptoris a rat Y2 receptor.

[0083] This invention also provides a method of screening drugs toidentify drugs which act as Y2 receptor agonists which comprisescontacting a cell transfected with and expressing nucleic acid encodinga Y2 receptor with a plurality of drugs under conditions permitting theactivation of a functional Y2 receptor response, and determining thosedrugs which activate such Y2 receptor, using a bioassay, such as asecond messenger assay, thereby identifying drugs which act as Y2receptor agonists. In one embodiment, the Y2 receptor is a human Y2receptor. In another embodiment the Y2 receptor is a rat Y2 receptor. Ina further embodiment, the Y2 receptor agonist is not previously known.

[0084] This invention provides a method of screening drugs to identifydrugs which act as agonists of a Y2 receptor which comprises preparing acell extract from cells transfected with and expressing nucleic acidencoding a Y2 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with a plurality of drugsunder conditions permitting the activation of a functional Y2 receptorresponse, and determining those drugs which activate such receptor,using a bioassay, such as a second messenger assay, thereby identifyingdrugs which act as Y2 receptor agonists. In one embodiment, the Y2receptor is a human Y2 receptor. In another embodiment, the Y2 receptoris a rat Y2 receptor. In a further embodiment, the Y2 receptor agonistis not previously known.

[0085] This invention also provides a method of screening drugs toidentify drugs which as Y2 receptor antagonists which comprisescontacting a cell transfected with and expressing nucleic acid encodinga Y2 receptor with a plurality of drugs in the presence of a known Y2receptor agonist such as NPY under conditions permitting the activationof a functional Y2 receptor response, and determining those drugs whichinhibit the activation of the receptor, using a bioassay, such as asecond messenger assay, thereby identifying drugs which act as Y2receptor antagonists. In one embodiment, the Y2 receptor is a human Y2receptor. In another embodiment, the Y2 receptor is a rat Y2 receptor.In a further embodiment, the Y2 receptor antagonist is not previouslyknown.

[0086] This invention provides a method of screening drugs to identifydrugs which act as Y2 receptor antagonists which comprises preparing acell extract from cells transfected with and expressing nucleic acidencoding a Y2 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with a plurality of drugs inthe presence of a known Y2 receptor agonist, such as NPY, underconditions permitting the activation of a functional Y2 receptorresponse, and determining those drugs which inhibit the activation ofthe receptor using a bioassay, such as a second messenger assay, therebyidentifying drugs which act as Y2 receptor antagonists. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor. In a furtherembodiment, the Y2 receptor antagonist is not previously known.

[0087] In one embodiment of the above described methods, the secondmessenger assay comprises measurement of intracellular cAMP. In anotherembodiment, the second messenger assay comprises measurement ofintracellular calcium mobilization.

[0088] The nucleic acid in the cells of the methods described above mayhave a coding sequence substantially the same as the coding sequencesshown in FIGS. 1, 8 and 9. Preferably, the mammalian cell is nonneuronalin origin. An example of a nonneuronal mammalian cell is an COS-7 cell.Other examples of a non-neuronal mammalian cell to be used forfunctional assays are 293 human embryonic kidney cells, mouse embryonicfibroblast NIH-3T3 cells and LM(tk-) cells. Drug candidates areidentified by choosing chemical compounds which bind with high affinityto the expressed Y2 receptor protein in transfected cells, usingradioligand binding methods well known in the art, examples of which areshown in the binding assays described herein. Drug candidates are alsoscreened for selectivity by identifying compounds which bind with highaffinity to the Y2 receptor but do not bind with high affinity to anyother NPY receptor subtype or to any other known receptor site. Becauseselective, high affinity compounds interact primarily with the target Y2receptor site after administration to the patient, the chances ofproducing a drug with unwanted side effects are minimized by thisapproach.

[0089] This invention provides a pharmaceutical composition comprisingan effective amount of a drug identified by the methods described aboveand a pharmaceutically acceptable carrier.

[0090] As used herein, an “effective amount” is an amount of the drugeffective to produce the desired result in a subject when administeredin accordance with the chosen regimen. Once the candidate drug has beenshown to be adequately bio-available following a particular route ofadministration, for example orally or by injection (adequate therapeuticconcentrations must be maintained at the site of action for an adequateperiod to gain the desired therapeutic benefit), and has been shown tobe non-toxic and therapeutically effective in appropriate diseasemodels, the drug may be administered to patients by that route ofadministration determined to make the drug bio-available, in anappropriate solid or solution formulation, to gain the desiredtherapeutic benefit.

[0091] This invention also provides a method of treating an abnormalityin a subject, wherein the abnormality is alleviated by activation of aY2 receptor which comprises administering to a subject an effectiveamount of the pharmaceutical composition described above, therebytreating the abnormality. In one embodiment, the Y2 receptor is a humanY2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

[0092] As used herein, the term “effective amount” means that amount ofa drug which is able to produce the desired result in a subject whenadministered in accordance with the chosen regimen. Typically, aneffective amount is an amount from about 0.01 mg per subject per day toabout 500 mg per subject per day. More typically this amount is anamount from about 0.1 mg per subject per day to about 60 mg per subjectper day. Most typically, this amount is an amount from about 1 mg persubject per day to about 20 mg per subject per day. Optimal dosages tobe administered may be determined by those skilled in the art, and willvary with the particular drug in use, the strength of the preparation,the mode of administration, and the advancement of the diseasecondition. Additional factors depending on the particular subject beingtreated will result in a need to adjust dosages, including subject age,weight, gender, diet, and time of administration.

[0093] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by activation of a Y2receptor which comprises administering to a subject an effective amountof a Y2 receptor agonist determined by the methods described above,thereby treating the abnormality. In one embodiment, the Y2 receptor isa human Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor.

[0094] This invention further provides a method of treating anabnormality in a subject, wherein the abnormality is alleviated bydecreasing the activity of a Y2 receptor which comprises administeringto a subject an effective amount of the pharmaceutical compositiondescribed above, thereby treating the abnormality. In one embodiment,the Y2 receptor is a human Y2 receptor. In another embodiment, the Y2receptor is a rat Y2 receptor.

[0095] This invention also provides a method of treating an abnormalityin a subject, wherein the abnormality is alleviated by decreasing theactivity of a Y2 receptor which comprises administering to the subjectan effective amount of a Y2 receptor antagonist determined by themethods described above, thereby treating the abnormality. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor.

[0096] This invention provides a nucleic acid probe comprising a nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with a unique sequence included within the sequence of anucleic acid molecule encoding a Y2 receptor, for example with a codingsequence included within the sequences shown in FIGS. 1, 8 and 9. Asused herein, the phrase “specifically hybridizing” means the ability ofa nucleic acid molecule to recognize a nucleic acid sequencecomplementary to its own and to form double-helical segments throughhydrogen bonding between complementary base pairs. As used herein, a“unique sequence” is a sequence specific to only the nucleic acidmolecules encoding the Y2 receptor. In one embodiment the Y2 receptor isa human Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor. Nucleic acid probe technology is well known to those skilledin the art who will readily appreciate that such probes may vary greatlyin length and may be labeled with a detectable label, such as aradioisotope or fluorescent dye, to facilitate detection of the probe.Detection of nucleic acid encoding Y2 receptors is useful as adiagnostic test for any disease process in which levels of expression ofthe corresponding Y2 receptor is altered. DNA probe molecules areproduced by insertion of a DNA molecule which encodes Y2 receptor orfragments thereof into suitable vectors, such as plasmids orbacteriophages, followed by insertion into suitable bacterial host cellsand replication and harvesting of the DNA probes, all using methods wellknown in the art. For example, the DNA may be extracted from a celllysate using phenol and ethanol, digested with restriction enzymescorresponding to the insertion sites of the DNA into the vector(discussed above), electrophoresed, and cut out of the resulting gel.Examples of such DNA molecules are shown in FIGS. 1, 8 and 9. The probesare useful for ‘in situ’ hybridization or in order to locate tissueswhich express this gene family, or for other hybridization assays forthe presence of these genes or their mRNA in various biological tissues.In addition, synthesized oligonucleotides (produced by a DNAsynthesizer) complementary to the sequence of a DNA molecule whichencodes a Y2 receptor are useful as probes for these genes, for theirassociated mRNA, or for the isolation of related genes by homologyscreening of genomic or cDNA libraries, or by the use of amplificationtechniques such as the Polymerase Chain Reaction. Synthesizedoligonucleotides as described may also be used to determine the cellularlocalization of the mRNA produced by the Y2 gene by in situhybridization.

[0097] This invention also provides a method of detecting expression ofa Y2 receptor by detecting the presence of mRNA coding for a Y2 receptorwhich comprises obtaining total mRNA from the cell using methods wellknown in the art and contacting the mRNA so obtained with a nucleic acidprobe comprising a nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a sequence included within thesequence of a nucleic acid molecule encoding the Y2 receptor underhybridizing conditions, and detecting the presence of mRNA hybridized tothe probe, thereby detecting the expression of the Y2 receptor by thecell. In one embodiment, the Y2 receptor is a human Y2 receptor. Inanother embodiment, the Y2 receptor is a rat Y2 receptor. Hybridizationof probes to target nucleic acid molecules such as mRNA moleculesemploys techniques well known in the art. In one possible means ofperforming this method, nucleic acids are extracted by precipitationfrom lysed cells and the mRNA is isolated from the extract using acolumn which binds the poly-A tails of the mRNA molecules. The mRNA isthen exposed to radioactively labelled probe on a nitrocellulosemembrane, and the probe hybridizes to and thereby labels complementarymRNA sequences. Binding may be detected by autoradiography orscintillation counting. However, other methods for performing thesesteps are well known to those skilled in the art, and the discussionabove is merely an example.

[0098] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to an mRNA molecule whichencodes a Y2 receptor so as to prevent translation of the mRNA molecule.The antisense oligonucleotide may have a sequence capable ofspecifically hybridizing with the cDNA molecule whose sequence is shownin FIG. 1, or with the genomic DNA molecule whose sequences are shown inFIGS. 8 and 9. A particular example of an antisense oligonucleotide isan antisense oligonucleotide comprising chemical analogues ofnucleotides.

[0099] This invention also provides a pharmaceutical compositioncomprising an amount of the oligonucleotide described above effective todecrease activity of a Y2 receptor by passing through a cell membraneand specifically hybridizing with mRNA encoding a Y2 receptor in thecell so as to prevent its translation and a pharmaceutically acceptablecarrier capable of passing through a cell membrane. The oligonucleotidemay be coupled to a substance which inactivates mRNA, such as aribozyme. The pharmaceutically acceptable carrier capable of passingthrough cell membranes may also comprise a structure which binds to areceptor specific for a selected cell type and is thereby taken up bycells of the selected cell type. The structure may be part of a proteinknown to bind a cell-type specific receptor, for example an insulinmolecule, which would target pancreatic cells. In one embodiment, the Y2receptor is a human Y2 receptor. In another embodiment, the Y2 receptoris a rat Y2 receptor. DNA molecules having coding sequencessubstantially the same as the coding sequences shown in FIGS. 1, 8 and 9may be used as the oligonucleotides of the pharmaceutical composition.

[0100] This invention also provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by decreasing theactivity of a Y2 receptor which comprises administering to the subjectan effective amount of the pharmaceutical composition described above,thereby treating the abnormality. In one embodiment, the Y2 receptor isa human Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor. Several examples of such abnormalities are hypertension,gastrointestinal disorders, epilepsy, sleep disorders, and cognitivedisorders, (58-80).

[0101] Antisense oligonucleotide drugs inhibit translation of mRNAencoding these receptors. Synthetic oligonucleotides, or other antisensechemical structures are designed to bind to mRNA encoding the Y2receptor and inhibit translation of mRNA and are useful as drugs toinhibit expression of Y2 receptor genes in patients. This inventionprovides a means to therapeutically alter levels of expression of Y2receptors by the use of a synthetic antisense oligonucleotide drug(SAOD) which inhibits translation of mRNA encoding these receptors.Synthetic oligonucleotides, or other antisense chemical structuresdesigned to recognize and selectively bind to mRNA, are constructed tobe complementary to portions of the nucleotide sequences shown in FIGS.1, 8, and 9 of DNA, RNA or of chemically modified, artificial nucleicacids. The SAOD is designed to be stable in the blood stream foradministration to patients by injection, or in laboratory cell cultureconditions, for administration to cells removed from the patient. TheSAOD is designed to be capable of passing through cell membranes inorder to enter the cytoplasm of the cell by virtue of physical andchemical properties of the SAOD which render it capable of passingthrough cell membranes (e.g. by designing small, hydrophobic SAODchemical structures) or by virtue of specific transport systems in thecell which recognize and transport the SAOD into the cell. In addition,the SAOD can be designed for administration only to certain selectedcell populations by targeting the SAOD to be recognized by specificcellular uptake mechanisms which binds and takes up the SAOD only withincertain selected cell populations. For example, the SAOD may be designedto bind to a receptor found only in a certain cell type, as discussedabove. The SAOD is also designed to recognize and selectively bind tothe target mRNA sequence, which may correspond to a sequence containedwithin the sequences shown in FIGS. 1, 8, and 9 by virtue ofcomplementary base pairing to the mRNA. Finally, the SAOD is designed toinactivate the target mRNA sequence by any of three mechanisms: 1) bybinding to the target mRNA and thus inducing degradation of the mRNA byintrinsic cellular mechanisms such as RNAse I digestion, 2) byinhibiting translation of the mRNA target by interfering with thebinding of translation-regulating factors or of ribosomes, or 3) byinclusion of other chemical structures, such as ribozyme sequences orreactive chemical groups, which either degrade or chemically modify thetarget mRNA. Synthetic antisense oligonucleotide drugs have been shownto be capable of the properties described above when directed againstmRNA targets (74,75). In addition, coupling of ribozymes to antisenseoligonucleotides is a promising strategy for inactivating target mRNA(76). An SAOD serves as an effective therapeutic agent if it is designedto be administered to a patient by injection, or if the patient's targetcells are removed, treated with the SAOD in the laboratory, and replacedin the patient. In this manner, an SAOD serves as a therapy to reducereceptor expression in particular target cells of a patient, in anyclinical condition which may benefit from reduced expression of Y2receptors.

[0102] This invention provides an antibody directed to a Y2 receptor,for example, a monoclonal antibody directed to an epitope of a Y2receptor present on the surface of a cell and having an amino acidsequence substantially the same as an amino acid sequence for a cellsurface epitope of the Y2 receptor included in the amino acid sequencesshown in FIGS. 2, 8 and 9 (Seq. I.D. Nos. 2, 4, and 6, respectively). Inone embodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor. Amino acid sequencesmay be analyzed by methods well known in the art to determine whetherthey produce hydrophobic or hydrophilic regions in the proteins whichthey build. In the case of cell membrane proteins, hydrophobic regionsare well known to form the part of the protein that is inserted into thelipid bilayer which forms the cell membrane, while hydrophilic regionsare located on the cell surface, in an aqueous environment. Thereforeantibodies to the hydrophilic amino acid sequences shown in FIGS. 2, 8,and 9 will probably bind to a surface epitope of a Y2 receptor, asdescribed. Antibodies directed to Y2 receptors may be serum-derived ormonoclonal and are prepared using methods well known in the art. Forexample, monoclonal antibodies are prepared using hybridoma technologyby fusing antibody producing B cells from immunized animals with myelomacells and selecting the resulting hybridoma cell line producing thedesired antibody. Cells such as COS-7 cells, LM(tk-) cells, NIH-3T3cells or 293 human embryonic cells comprising DNA encoding the Y2receptor and thereby expressing the Y2 receptor may be used asimmunogens to raise such an antibody. Alternatively, synthetic peptidesmay be prepared using commercially available machines and the amino acidsequences shown in FIGS. 2, 8, and 9 (Seq. I.D. Nos. 2, 4, and 6,respectively). As a still further alternative, DNA, such as a cDNA or afragment thereof, may be cloned and expressed and the resultingpolypeptide recovered and used as an immunogen. These antibodies areuseful to detect the presence of Y2 receptors encoded by the isolatedDNA, or to inhibit the function of the receptors in living animals, inhumans, or in biological tissues or fluids isolated from animals orhumans.

[0103] This invention provides a pharmaceutical composition whichcomprises an amount of an antibody directed to a Y2 receptor effectiveto block binding of ligands to the Y2 receptor, and a pharmaceuticallyacceptable carrier. A monoclonal antibody directed to an epitope of a Y2receptor present on the surface of a cell and having an amino acidsequence substantially the same as an amino acid sequence for a cellsurface epitope of the Y2 receptor included in the amino acid sequencesshown in FIGS. 2, 8 and 9 are useful for this purpose.

[0104] This invention also provides a method of treating an abnormalityin a subject, wherein the abnormality is alleviated by decreasing theactivity of a Y2 receptor which comprises administering to the subjectan amount of the pharmaceutical composition described above effective toblock binding of ligands to the Y2 receptor, thereby treating theabnormality. In a one embodiment, the Y2 receptor is a human Y2receptor. In another embodiment, the Y2 receptor is a rat Y2 receptor.Binding of the antibody to the receptor prevents the receptor fromfunctioning, thereby neutralizing the effects of activity of thereceptor. The monoclonal antibodies described above are both useful forthis purpose. Some examples of such abnormalities are hypertension,gastrointestinal disorders, epilepsy, sleep disorders, and cognitivedisorders (58-72).

[0105] This invention provides a method of detecting the presence of aY2 receptor on the surface of a cell which comprises contacting the cellwith an antibody directed to the Y2 receptor, under conditionspermitting binding of the antibody to the receptor, and detecting thepresence of the antibody bound to the cell, thereby detecting thepresence of a Y2 receptor on the surface of the cell. Such a method isuseful for determining whether a given cell is defective in expressionof Y2 receptors on the surface of the cell. Bound antibodies aredetected by methods well known in the art, for example by bindingfluorescent markers to the antibodies and examining the cell sampleunder a fluorescence microscope to detect fluorescence on a cellindicative of antibody binding. The monoclonal antibodies describedabove are useful for this purpose.

[0106] This invention provides a transgenic nonhuman mammal expressingnucleic acid encoding a Y2 receptor. In one embodiment, the Y2 receptoris a human Y2 receptor. In another embodiment, the Y2 receptor is a ratY2 receptor. This invention also provides a transgenic nonhuman mammalcomprising a homologous recombination knockout of the native Y2receptor. This invention also provides a transgenic nonhuman mammalwhose genome comprises antisense nucleic acid complementary to nucleicacid encoding a Y2 receptor so placed as to be transcribed intoantisense mRNA which is complementary to mRNA encoding a Y2 receptor andwhich hybridizes to mRNA encoding a Y2 receptor thereby reducing itstranslation. The nucleic acid may additionally comprise an induciblepromoter or additionally comprise tissue specific regulatory elements,so that expression can be induced, or restricted to specific cell types.Examples of nucleic acid are DNA or cDNA molecules having a codingsequence substantially the same as the coding sequences shown in FIGS.1, 8, and 9. An example of a transgenic animal is a transgenic mouse.Examples of tissue specificity-determining regions are themetallothionein promotor (77) and the L7 promotor (78).

[0107] Animal model systems which elucidate the physiological andbehavioral roles of Y2 receptors are produced by creating transgenicanimals in which the activity of a Y2 receptor is either increased ordecreased, or the amino acid sequence of the expressed Y2 receptorprotein is altered, by a variety of techniques. Examples of thesetechniques include: 1) Insertion of normal or mutant versions of nucleicacid encoding a Y2 receptor or homologous animal versions of thesegenes, by microinjection, retroviral infection or other means well knownto those skilled in the art, into appropriate fertilized embryos inorder to produce a transgenic animal (79). 2) Homologous recombination(80, 81) of mutant or normal, human or animal versions of these geneswith the native gene locus in transgenic animals to alter the regulationof expression or the structure of these Y2 receptors. The technique ofhomologous recombination is well known in the art. It replaces thenative gene with the inserted gene and so is useful for producing ananimal that cannot express native receptor but does express, forexample, an inserted mutant receptor, which has replaced the nativereceptor in the animal's genome by recombination, resulting inunderexpression of the receptor. Microinjection adds genes to thegenome, but does not remove them, and so is useful for producing ananimal which expresses its own and added receptors, resulting inoverexpression of the receptor. One means available for producing atransgenic animal, with a mouse as an example, is as follows: Femalemice are mated, and the resulting fertilized eggs are dissected out oftheir oviducts. The eggs are stored in an appropriate medium such as M2medium (79). DNA or cDNA encoding a Y2 receptor is purified from avector (such as plasmid pcEXV-hY2, pcEXV-rY2a or pcEXV-rY2b describedabove) by methods well known in the art. Inducible promoters may befused with the coding region of the nucleic acid to provide anexperimental means to regulate expression of the trans-gene.Alternatively, or in addition, tissue specific regulatory elements maybe fused with the coding region to permit tissue-specific expression ofthe trans-gene. The nucleic acid, in an appropriately buffered solution,is put into a microinjection needle (which may be made from capillarytubing using a pipet puller) and the egg to be injected is put in adepression slide. The needle is inserted into the pronucleus of the egg,and the nucleic acid solution is injected. The injected egg is thentransferred into the oviduct of a pseudopregnant mouse (a mousestimulated by the appropriate hormones to maintain pregnancy but whichis not actually pregnant), where it proceeds to the uterus, implants,and develops to term. As noted above, microinjection is not the onlymethod for inserting nucleic acid into the egg cell, and is used hereonly for exemplary purposes.

[0108] Since the normal action of receptor-specific drugs is to activateor to inhibit the receptor, the transgenic animal model systemsdescribed above are useful for testing the biological activity of drugsdirected against these Y2 receptors even before such drugs becomeavailable. These animal model systems are useful for predicting orevaluating possible therapeutic applications of drugs which activate orinhibit these Y2 receptors by inducing or inhibiting expression of thenative or trans-gene and thus increasing or decreasing activity ofnormal or mutant Y2 receptors in the living animal. Thus, a model systemis produced in which the biological activity of drugs directed againstthese Y2 receptors are evaluated before such drugs become available. Thetransgenic animals which over or under produce the Y2 receptor indicateby their physiological state whether over or under production of the Y2receptor is therapeutically useful. It is therefore useful to evaluatedrug action based on the transgenic model system. One use is based onthe fact that it is well known in the art that a drug such as anantidepressant acts by blocking neurotransmitter uptake, and therebyincreases the amount of neurotransmitter in the synaptic cleft. Thephysiological result of this action is to stimulate the production ofless receptor by the affected cells, leading eventually to decreasedactivity. Therefore, an animal which has decreased receptor activity isuseful as a test system to investigate whether the actions of such drugswhich result in decreased activity are in fact therapeutic. Another useis that if increased activity is found to lead to abnormalities, then adrug which down-regulates or acts as an antagonist to a Y2 receptor isindicated as worth developing, and if a promising therapeuticapplication is uncovered by these animal model systems, activation orinhibition of the Y2 receptor is achieved therapeutically either byproducing agonist or antagonist drugs directed against these Y2receptors or by any method which increases or decreases the activity ofthese Y2 receptors in humans or other mammals.

[0109] This invention provides a method of determining the physiologicaleffects of expressing varying levels of Y2 receptors which comprisesproducing a transgenic nonhuman animal whose levels of Y2 receptorexpression are varied by use of an inducible promoter which regulates Y2receptor expression. This invention also provides a method ofdetermining the physiological effects of expressing varying levels of Y2receptors which comprises producing a panel of transgenic nonhumananimals each expressing a different amount of Y2 receptor. In oneembodiment, the Y2 receptor is a human Y2 receptor. In anotherembodiment, the Y2 receptor is a rat Y2 receptor. Such animals may beproduced by introducing different amounts of nucleic acid encoding a Y2receptor into the oocytes from which the transgenic animals aredeveloped.

[0110] This invention also provides a method for identifying a Y2receptor antagonist capable of alleviating an abnormality is a subject,wherein the abnormality is alleviated by decreasing the acitivity of aY2 receptor which comprises administering the antagonist to a transgenicnonhuman mammal described above and determining whether the antagonistalleviates the physical and behavioral abnormalities displayed by thetransgenic nonhuman mammal as a result of the activity of a Y2 receptor,thereby identifying a Y2 antagonist. In one embodiment, the Y2 receptoris a human Y2 receptor, In another embodiment, the Y2 receptor is a ratY2 receptor. This invention further provides an antagonist identified bythe method described above. Examples of nucleic acid molecules are DNAor cDNA molecules having a coding sequence substantially the same as thecoding sequences shown in FIGS. 1, 8, and 9.

[0111] This invention provides a pharmaceutical composition comprisingan amount of the antagonist described supra effective to alleviate anabnormality wherein the abnormality is alleviated by decreasing theactivity of a Y2 receptor and a pharmaceutically acceptable carrier.

[0112] This invention further provides a method for treating anabnormality in a subject wherein the abnormality is alleviated bydecreasing the activity of a Y2 receptor which comprises administeringto the subject an effective amount of the pharmaceutical compositiondescribed above, thereby treating the abnormality.

[0113] This invention provides a method for identifying a Y2 receptoragonist capable of alleviating an abnormality wherein the abnormality isalleviated by activation of a Y2 receptor which comprises administeringthe agonist to the transgenic nonhuman mammal described above anddetermining whether the agonist alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal, therebyidentifying a Y2 receptor agonist. In one embodiment, the Y2 receptor isa human Y2 receptor. In another embodiment, the Y2 receptor is a rat Y2receptor. This invention further provides an agonist identified by themethod described above.

[0114] This invention also provides a pharmaceutical compositioncomprising an effective amount of a Y2 receptor agonist identified bythe method described above and a pharmaceutically acceptable carrier.

[0115] This invention further provides a method for treating anabnormality in a subject wherein the abnormality is alleviated byactivation of a Y2 receptor which comprises administering to the subjectan effective amount of the pharmaceutical composition described above,thereby treating the abnormality.

[0116] This invention provides a method for diagnosing a predispositionto a disorder associated with the expression of a specific Y2 receptorallele which comprises: a) obtaining nucleic acid of subjects sufferingfrom the disorder; b) performing a restriction digest of the nucleicacid with a panel of restriction enzymes; c) electrophoreticallyseparating the resulting nucleic acid fragments on a sizing gel; d)contacting the resulting gel with a nucleic acid probe capable ofspecifically hybridizing to nucleic acid encoding a Y2 receptor andlabelled with a detectable marker; e) detecting labelled bands whichhave hybridized to the nucleic acid encoding a Y2 receptor labelled witha detectable marker to create a unique band pattern specific to thenucleic acid of subjects suffering from the disorder; f) preparingnucleic acid obtained for diagnosis by steps a-e; and g) comparing theunique band pattern specific to the nucleic acid of subjects sufferingfrom the disorder from step e and the nucleic acid obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and thereby to diagnose predisposition to the disorder if thepatterns are the same. This method may also be used to diagnose adisorder associated with the expression of a specific Y2 receptorallele. In one embodiment, the Y2 receptor is a human Y2 receptor. Inanother embodiment, the Y2 receptor is a rat Y2 receptor.

[0117] This invention provides a method of preparing the isolated,purified Y2 receptor which comprises a) constructing a vector adaptedfor expression in a cell which comprises the regulatory elementsnecessary for the expression of nucleic acid in the cell operativelylinked to the nucleic acid encoding a Y2 receptor as to permitexpression thereof., wherein the cell is selected form the groupconsisting of bacterial cells, yeast cells, insect cells and mammaliancells; b) inserting the vector of step (a) in a suitable host cell; c)incubating the cells of step (b) under conditions allowing theexpression of a Y2 receptor; d) recovering the receptor so produced; ande) purifying the receptor so recovered. An example of an isolated Y2receptor is an isolated protein having substantially the same amino acidsequence as the amino acid sequences shown in FIGS. 2, 8 and 9. Forexample, cells can be induced to express receptors by exposure tosubstances such as hormones. The cells can then be homogenized and thereceptor isolated from the homogenate using an affinity columncomprising, for example, PYY or NPY or another substance which is knownto bind to the receptor. The resulting fractions can then be purified bycontacting them with an ion exchange column, and determining whichfraction contains receptor activity or binds anti-receptor antibodies.

[0118] The above described method for preparing a Y2 receptor usesrecombinant DNA technology methods well known in the art. For example,isolated nucleic acid encoding Y2 receptor is inserted in a suitablevector, such as an expression vector. A suitable host cell, such as abacterial cell, or a eukaryotic cell such as a yeast cell, istransfected with the vector. Y2 receptor is isolated from the culturemedium by affinity purification or by chromatography or by other methodswell known in the art.

[0119] This invention identifies for the first time a new receptorprotein, its amino acid sequence, its human gene and its rat homologue.Furthermore, this invention describes a previously unrecognized group ofreceptors within the definition of a Y2 receptor. The information andexperimental tools provided by this discovery are useful to generate newtherapeutic agents, and new therapeutic or diagnostic assays for thisnew receptor protein, its associated mRNA molecule or its associatedgenomic DNA. The information and experimental tools provided by thisdiscovery will be useful to generate new therapeutic agents, and newtherapeutic or diagnostic assays for this new receptor protein, itsassociated mRNA molecule, or its associated genomic DNA.

[0120] Specifically, this invention relates to the first isolation of ahuman genomic clone encoding a Y2 receptor. A new human gene for thereceptor identified herein as Y2 has been identified and characterized.In addition, the human Y2 receptor has been expressed in 293 humanembryonic kidney cells. The pharmacological binding properties of theprotein encoded have been determined, and these binding propertiesclassify this protein as a novel human NPY/PYY receptor which wedesignate as a human Y2 receptor. Mammalian cell lines expressing thishuman Y2 receptor at the cell surface have been constructed, thusestablishing the first well-defined, cultured cell lines with which tostudy this Y2 receptor.

[0121] This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0122] Experimental Details

[0123] cDNA Cloning

[0124] Total RNA was prepared by a modification of the guanidinethiocyanate method (13), from 6 grams of human hippocampus. Poly A+RNAwas purified with a FastTrack kit (Invitrogen Corp., San Diego, Calif.).Double stranded (ds) cDNA was synthesized from 4 μg of poly A⁺ RNAaccording to Gubler and Hoffman (14), except that ligase was omitted inthe second strand cDNA synthesis. The resulting DS cDNA was ligated toBstxI/EcoRI adaptors (Invitrogen Corp.), the excess of adaptors wasremoved by chromatography on Sephacryl®500 HR (Pharmacia-LKB) and theds-cDNA size selected by chromatography on Sephacryl® 1000(Pharmacia-LKB). High molecular weight fractions were ligated inpcEXV.BS (An Okayama and Berg expression vector) cut by BstxI asdescribed by Aruffo and Seed (15). The ligated DNA was electroporated inE. coli MC 1061 (Gene Pulser, Biorad). A total of 2.2×10⁶ independentclones with an insert mean size of 3 kb could be generated. The librarywas plated on Petri dishes (Ampicillin selection) in pools of 0.4 to1.2×10⁴ independent clones. After 18 hours amplification, the bacteriafrom each pool were scraped, resuspended in 4 mL of LB media and 1.5 mLprocessed for plasmid purification by the alkali method (16). 1 mLaliquots of each bacterial pool were stored at −85° C. in 20% glycerol.

[0125] Isolation of a cDNA Clone Encoding a Human Hippocampal Y2Receptor.

[0126] DNA from pools of ≈5000 independent clones was transfected intoCOS-7 cells by a modification of the DEAE-dextran procedure (17). COS-7cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplementedwith 10% fetal calf serum, 100 U/mL of penicillin, 100 μg/mL ofstreptomycin, 2 mM L-glutamine (DMEM-C) at 37° C. in 5% CO₂. The cellswere seeded one day before transfection at a density of 30,000 cells/cm²in 6 well plates (Becton Dickinson, Lincoln Park, N.J.). On the nextday, cells were washed twice with Phosphate Buffer Saline (PBS), 400 μlof transfection cocktail was added containing {fraction (1/10)} of theDNA from each pool and DEAE-dextran (500 μg/mL) in PBS. After a 30 min.incubation at 37° C., 1.6 mL of chloroquine (80 μM in DMEM-C) was addedand the cells incubated a further 2.5 hours at 37° C. The media wasaspirated from each well and 1 mL of 10% DMSO in DMEM-C added. After 2.5min. incubation at room temperature, the media was aspirated, each wellwashed once with 1 mL PBS and the cells incubated 24 hours in DMEM-C.The cells were then trypsinized and seeded on Lab-Tek®chamber slides (1chamber, Permanox slide from Nunc Inc., Naperville, Ill.), incubated in2 ml DMEM-C for another 24 hours and the binding assay was performed onthe slides.

[0127] After two washes with PBS, positive pools were identified byincubating the cells with 1 nM (3×10⁶ cpm per slide) of porcine[¹²⁵I]-PYY (New England Nuclear; specific activity=2200Ci/mmol) in 20 mMHepes-NaOH pH 7.4, CaCl₂ 1.26 mM, MgSO₄ 0.81 mM, KHzPO₄ 0.44 mM, KCl5.4, NaCl 10 mM, 0.1% bovine serum albumin, 0.1% bacitracin for 1 hourat room temperature. After six washes (five seconds each) in bindingbuffer without ligand, the monolayers were fixed in 2.5% glutaraldehydein PBS for five minutes, washed twice two minutes in PBS, dehydrated inethanol baths for two minutes each (70, 80, 95, 100%) and air dried.

[0128] The slides were then dipped in 100% photoemulsion (Kodak® typeNTB2) at 42° C. and exposed in the dark for 48 hours at 4° C. in lightproof boxes containing drierite. Slides were developed for three minutesin Kodak®D19 developer (32 g/l of water), rinsed in water, fixed inKodak®fixer for 5 minutes, rinsed in water, air dried and mounted withAqua Mount® (Lerner Laboratories, Pittsburgh, Pa.). Slides were screenedat 25× total magnification.

[0129] A single clone, CG-13, was isolated by sib selection as described(18). DS-DNA was sequenced with a Sequenase® kit (US Biochemical,Cleveland, Ohio) according to the manufacturer. Nucleotide and peptidesequences analysis were performed with GCG programs (Genetics Computergroup, Madison, Wis.).

[0130] Northern Blot

[0131] A multiple tissue Northern blot (MTN blot, Contech, Palo Alto,Calif.) carrying mRNA purified from various human brain areas washybridized at high stringency according to the manufacturer'sspecifications. The probe was a 1.15 kb DNA fragment corresponding tothe entire coding region of the human Y2 receptor as shown in FIG. 10.

[0132] Southern Blot:

[0133] A Southern blot (Geno-Blot, Clontech, Palo Alto, Calif.)containing human genomic DNA cut with five different enzymes (8 μg DNAper lane) was hybridized at high stringency according to themanufacturer's specifications. The probe was a DNA fragmentcorresponding to the TM1-TM5 coding region of the human Y2 receptor, asshown in FIG. 11.

[0134] Cloning and Expression of Two Isoforms of the Rat NPY/PYY (Y2)Receptor

[0135] To obtain the rat homologue of the human NPY/PYY (Y2) receptor,we designed and synthesized oligonucleotide probes derived from thenucleotide sequences corresponding approximately to the transmembrane(TM) regions of the amino acid sequence of the human Y2 receptor (TM1-7) as shown in FIG. 11. The overlapping oligomers used were asfollows:

[0136] (TM1: nts. #190-257, (+)strand/5′-CAAGTTGTTCTCATATTGGCCTACTGCTCCATCATCTTGCTTGGGGTAAT-3′ (Seq.I.D. No. 7) and (−)strand/5′-ATCACCACATGGATCACCAAGGAGTTGCCAATTACCCCAAGCAAGATGAT-3′ (Seq.I.D. No. 8)

[0137] TM2: nts. #301-370, (+)strand/5′-TTTTTCATTGCCAATCTGGCTGTGGCAGATCTTTTGGTGAACACT-3′ (Seq. I.D.No. 9) and (−)strand/5′-AGGTAAGAGTGAACGGTAGACACAGAGTGTTCACCAAAAGATCTG-3′ (Seq. I.D.No. 10).

[0138] TM3: nts. #411-480, (+)strand/5′-CCACCTGGTGCCCTATGCCCAGGGCCTGGCAGTACAAGTATCCAC-3′ (Seq. I.D.No. 11) and (−)strand/5′-CAGGGCAATTACTGTCAAGGTGATTGTGGATACTTGTACTGCCAG-3′ (Seq. I. D.No. 12).

[0139] TM4: nts. #531-600, (+)strand/5′-AATCAGCTTCCTGATTATTGGCTTGGCCTGGGGCATCAGTGCCCT-3′ (Seq. I.D.No. 13) and (−)strand/5′-GAAGATGGCCAGGGGACTTGCCAGCAGGGCACTGATGCCCCAGGC-3′ (Seq. I.D.No. 14)

[0140] TM5: nts. #691-760, (+)strand/5′-ACTGTCTATAGTCTTTCTTCCTTGTTGATCTTGTATGTTTTGCCT-3′ (Seq. I.D.No. 15) and (−)strand/5′-TGTAGGAAAATGATATAATGCCCAGAGGCAAAACATACAAGATCA-3′ (Seq. I.D.NO. 16)

[0141] TM6: nts. #850-919, (+)strand/5′-CTGGTGTGTGTGGTGGTGGTGTTTGCGGTCAGCTGGCTGCCTCTC-3′ (Seq. I.D.No. 17) and (−)strand/5′-TGTCAACGGCAAGCTGGAAGGCATGGAGAGGCAGCCAGCTGACCG-3′ (Seq. I.D.No. 18)

[0142] TM7: NTS. #955-1028, (+)strand/5′-CTCATCTTCACAGTGTTCCACATCATCGCCATGTGCTCCACTTTTGC-3′ (Seq. I.D.No. 19) and (−)strand/5′-TTCATCCAGCCATAGAGAAGGGGATTGGCAAAAGTGGAGCACATGGC-3′ (Seq. I.D.No. 20).

[0143] The probes were labeled with [³²P]-ATP and [³²p]-CTP by synthesiswith the large fragment of DNA polymerase.

[0144] Hybridization was performed at 40° C. in a solution containing25% formamide, 10% dextran sulfate, 5×SSC (1×SSC is 0.15 M sodiumchloride, 0.015 M sodium citrate), 1× Denhardt's (0.02%polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and100 μg/ml of sonicated salmon sperm DNA. The filters were washed at 40°C. in 0.1×SSC containing 0.1% sodium dodecyl sulfate (SDS) and exposedat −70° C. to Kodak® XAR film in the presence of one intensifyingscreen. Lambda phage hybridizing to the probes were plaque purified bysuccessive plating and rescreening. A genomic clone hybridizing with sixout of seven TM probes, designated rs5a, was isolated using this method.A 4.0 kb EcoRI fragment of rs5a was subcloned into the eukaryoticexpression vector EXJ.RH modified from pcEXV-3 (73) for sequenceanalysis and expression studies. The nucleotide sequence of the fragmentin EXJ.RH was analyzed on both strands by the Sanger dideoxy nucleotidechain-termination method (82) using Sequenase® (U.S. Biochemical Corp.,Cleveland, Ohio).

[0145] A second genomic clone, termed rs26a, was also isolated using thehybridization conditions described above and exhibited the samehybridization profile with TM probes. In contrast with rs5a, however,rs26a contained an internal EcoRI restriction enzyme site not present inthe other clone. To further investigate potential differences betweenthe two clones, a 3.9 kb SalI/KpnI fragment of rs26a was subcloned intothe expression vector EXJ.HR for sequence analysis and expressionstudies. The nucleotide sequence of the fragment was analyzed on bothstrands by the Sanger dideoxy nucleotide chain-termination method asdescribed above.

[0146] Cell Culture

[0147] COS-7 cells were grown on 150 mm plates in Dulbecco's ModifiedEagle Medium (DMEM) with supplements (10% bovine calf serum, 4 mMglutamine, 100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells were trypsinized and split 1:6 every3-4 days.

[0148] Human embryonic kidney cells 293 cells were grown on 150 mmplates in Dulbecco's Modified Eagle Medium (DMEM) with supplements (10%bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of 293 cells weretrypsinized and split 1:6 every 3-4 days. Mouse embryonic fibroblastNIH-3T3 cells were grown on 150 mm plates in Dulbecco's Modified EagleMedium (DMEM) with supplements (10% bovine calf serum, 4 mM glutamine,100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5% CO₂. Stockplates of NIH-3T3 cells were trypsinized and split 1:15 every 3-4 days.

[0149] SK—N—Be(2) human neuroblastoma cells were grown similarly in 225cm² flasks using 50% Eagle's Modified Essential Media, 50% Ham'sNutrient Mixture F-12, 15% fetal bovine serum, 2 mM glutamine, 100units/ml penicillin/80 units/ml streptomycin, and 1% non-essential aminoacids. Stock flasks of SK—N—Be(2) cells were trypsinized and split 1:10every 7 days.

[0150] DNA Transfection for Pharmacological Characterization

[0151] All cloned receptor subtypes studied (human Y1, human Y2, humanY4, rat Y2a and rat Y2b) were transiently transfected into COS-7 cellsby the DEAE-dextran method, using 1 μg of DNA/10⁶ cells (17). The cDNAcorresponding to the cloned Y4 receptor was disclosed in U.S. patentapplication Ser. No. 08/176,412 filed on Dec. 28, 1993, currentlypending.

[0152] Membrane Preparation

[0153] Membranes were harvested from COS-7 cells 48 hours aftertransfection and from SK—N—Be(2) seven days after splitting. Adherentcells were washed twice in ice-cold phosphate buffered saline (138 mMNaCl, 8.1 mM Na₂HPO₄, 2.5 mM KCl, 1.2 mM KH₂PO₄, 0.9 mM CaCl₂, 0.5 mMMgCl₂, pH 7.4) and lysed by sonication in ice-cold hypotonic buffer (20mM Tris-HCl, 5 mM EDTA, pH 7.7). Large particles and debris were clearedby low speed centrifugation (200×g, 20 min, 4° C.). Membranes werecollected from the supernatant fraction by high speed centrifugation(32,000×g, 18 min, 4° C.), washed with ice-cold hypotonic buffer, andcollected again by high speed centrifugation (32,000×g, 18 min, 4° C.).The final membrane pellet was resuspended by sonication into a smallvolume (˜500 μl) of ice-cold binding buffer (10 mM NaCl, 20 mM HEPES,0.22 mM KH₂PO₄, 1.26 mM CaCl₂, 0.81 mM MgSO₄, pH 7.4). Proteinconcentration was measured by the Bradford method (19) using Bio-RadReagent, with bovine serum albumin as a standard.

[0154] Radioligand Binding to Membrane Suspensions

[0155] Membrane suspensions were diluted in binding buffer supplementedwith 0.1% bovine serum albumin and 0.1% bacitracin to yield membraneprotein concentrations of ˜0.02 mg/ml for human Y1 receptors, ˜0.003mg/ml for CG-13 receptors, and ˜0.25 mg/ml for SK—N—Be(2) (under theseassay conditions, non-specific binding of ¹²⁵I-PYY to membranes was lessthan 10%). ¹²⁵I-PYY and non-labeled peptide competitors were alsodiluted to desired concentrations in supplemented binding buffer.Individual samples were then prepared in 96-well polypropylenemicroliter plates by mixing membrane suspensions (200 ul), ¹²⁵I-PYY (25ul), and non-labeled peptides or supplemented binding buffer (25 ul).Samples were incubated in a 30° C. water bath with constant shaking for120 min. Incubations were terminated by filtration over Whatman®GF/Cfilters pre-coated with 0.5% polyethyleneimine and air-dried beforeuse). Filter-trapped membranes were counted for ¹²⁵I in a gamma counter.Non-specific binding was defined by 100 nM human NPY. Specific bindingin time course and competition studies was typically 80%; mostnon-specific binding was associated with the filter. Binding data wereanalyzed using nonlinear regression and statistical techniques availablein the GraphPAD®InPlot package (San Diego, Calif.).

[0156] Creation of a Stably Expressing Cell Line

[0157] pcEXV-hY2 DNA was transfected into the 293 human embryonic kidneycell line by the calcium phosphate transfection method. The 293 cellswere grown in minimal essential medium (MEM) with Hank's salts, plus 2mM glutamine, 100 international units of penicillin, streptomycin at 100ug/ml, and 10% fetal calf serum, in 5% CO₂ at 37° C. Stably transfectedcells were selected for two weeks in media containing G-148 (1 mg/ml)and screened for the ability to bind ¹²⁵I-PYY. Several clones wereselected based on preliminary measurements of cell density. One positiveclone, designated 293-hY2-10, was chosen for further characterization inbinding and functional assays. This clone displayed saturable binding of¹²⁵I-porcine PYY in membrane preparations: B_(max)=880 fmol/mg membraneprotein, K_(d)=3 pM, (n=3). When incubated with various concentrationsof human PYY, it elicited a concentration-dependent inhibition offorskolin-stimulated cAMP accumulation as determined byradioimmunoassay. Clone 293-hY2-10 also elicited aconcentration-dependent increase in free intracellular calcium asdetermined by Fura-2 florescence. The calcium response, which probablyreflects mobilization of intracellular calcium stores, was inhibited bypretreatment of cells with pertussis toxin. EC₅₀ values for both thecAMP and the calcium response are currently under investigation.

[0158] pcEXV-hY2 DNA was also transfected into the mouse embryonicNIH-3T3 cell line using the methods described above to create anothercell line stably expressing human Y2 receptors. A clone designatedN-hY2-5 was selected and characterized as above.

[0159] Tissue Localization and Gene Expression: Reverse TranscriptasePCR

[0160] Human tissues obtained from National Disease Research Interchangewere homogenized and total RNA extracted using guanidineisothiocyanate/CsCl cushion method. RNA was treated with DNase to removeany contaminating genomic DNA. cDNA was prepared from total RNA withrandom hexanucleotide primers using the reverse transcriptaseSuperscript II (BRL, Gaithersburg, Md.). An aliquot of the first strandcDNA (250 ng of total RNA) was amplified in a 50 μl PCR reaction mixture(200 μM dNTPs final concentration) containing 1.2U of Taq polymerase inthe buffer supplied by the manufacturer (Perkin-Elmer Corporation), and1 μM of primers, using a program consisting of 30 cycles of 94° C./2′,68° C./2′, and 72° C./3′, with a pre- and post-incubation of 95° C./5′and 72° C./10′, respectively. PCR primers for human Y2 were designedagainst the human Y2 sequence in the third intracellular loop andcarboxyl terminal regions: 5′-GGGAGTATTCGCTGATTGAGATCAT-3′ (SEQ. I.D.No. 21) and 5′-GCCTTGAATGTCACGGACACCTC-3′ (SEQ. I.D. No. 22),respectively.

[0161] The PCR products were run on a 1.5% agarose gel and transferredto charged nylon membranes (Zetaprobe GT, BioRad), and analyzed asSouthern blots. Hybridization probes corresponding to the receptorregion flanked by PCR primers were prepared(5′-CTGATGGTAGTGGTCATTTGCAGCTCCAGGACTGACATGGTTCTT-3′) (SEQ. I.D. No. 23)and pre-screened for the absence of cross-reactivity with human Y1 andY4 receptor subtypes. Filters were hybridized with the phosphorylatedprobes and washed under high stringency. Labeled PCR products werevisualized on X-ray film. Similar PCR and Southern blot analyses wereconducted with primers and probe directed to the housekeeping gene,glyceraldehyde-3-phosphate dehydrogenase (Clontech, Palo Alto, Calif.),and demonstrated that equal amounts of cDNA from the different tissueswere being assayed for Y2 receptor expression.

[0162] Localization of NPY Y2 Messenger RNA in the Rat Central NervousSystem

[0163] The distribution of NPY Y2 mRNA in the rat brain was determinedusing in situ hybridization histochemistry. Male Sprague-Dawley ratswere euthanized with CO₂, decapitated and the brains rapidly removed andfrozen in isopentane. Coronal sections were cut at 11 μm on a cryostatand thaw-mounted onto poly-L-lysine coated slides and stored at −80° C.until use. Prior to hybridization, tissues were fixed in 4%paraformaldehyde, treated with 5 mM dithiothreitol, acetylated in 0.1 Mtriethanolamine containing 0.25% acetic anhydride, delipidated withchloroform, and dehydrated in graded ethanols.

[0164] The oligonucleotide probes employed to characterize thedistribution of the NPY Y2 mRNA were synthesized using a Cyclone PlusDNA synthesizer (Milligen/Biosearch) and gel-purified. The probes usedand their sequences are given in Table 7. Probe specificity wasestablished by performing the in situ hybridization protocol describedbelow on cells transfected with the rat NPY Y2 DNA (supra), or onnontransfected control cells. In addition, both sense and antisenseprobes were employed on cells and rat tissues.

[0165] Probes were 3′-end labeled with ³⁵S-dATP (1200 Ci/mmol, NewEngland Nuclear, Boston, Mass.) to a specific activity of 109 dpm/μgusing terminal deoxynucleotidyl transferase (Boehringer Mannheim;Indianapolis, Ind.). The radiolabeled probes were purified on Biospin® 6chromatography columns (Bio-Rad; Richmond, Calif.), and diluted inhybridization buffer to a concentration of 1.5×10⁴ cpm/μl. Thehybridization buffer consisted of 50% formamide, 4× sodium citratebuffer (1×SSC=0.15 M NaCl and 0.015 M sodium citrate), 1× Denhardt'ssolution (0.2% polyvinylpyrrolidine, 0.2% Ficoll, 0.2% bovine serumalbumin), 50 mM dithiothreitol, 0.5 mg/ml salmon sperm DNA, 0.5 mg/mlyeast tRNA, and 10% dextran sulfate. One hundred μl of the diluted probewas applied to each section, which was then covered with a Parafilmcoverslip. Hybridization was carried out overnight in humid chambers at40 to 55° C. The following day the sections were washed in two changesof 2×SSC for one hour at room temperature, in 0.1×SSC for 30 min at50-60° C., and finally in 0.1×SSC for 30 min at room temperature.Tissues were dehydrated in graded ethanols and apposed to Kodak® XAR-5film for 3 days to 6 weeks at −20° C., then dipped in Kodak® NTB3autoradiography emulsion diluted 1:1 with 0.2% glycerol water. Afterexposure at 4° C. for 2 to 8 weeks, the slides were developed in Kodak®D-19 developer, fixed, and counterstained with hematoxylin and eosin.

[0166] Functional Assay: Radioimmunoassay of cAMP

[0167] Stably transfected cells were seeded into 96-well microliterplates and cultured until confluent. To reduce the potential forreceptor desensitization, the serum component of the media was reducedto 1.5% for 4 to 16 hours before the assay. Cells were washed in Hank'sbuffered saline, or HBS (150 mM NaCl, 20 mM HEPES, 1 mM CaCl₂, 5 mM KCl,1 mM MgCl₂, and 10 mM glucose) supplemented with 0.1% bovine serumalbumin plus mM theophylline and pre-equilibrated in the same solutionfor 20 min at 37° C. in 5% CO₂. Cells were then incubated 5 min with 10μM forskolin and various concentrations of receptor-selective ligands.The assay was terminated by the removal of HBS and acidification of thecells with 100 mM HCl. Intracellular cAMP was extracted and quantifiedwith a modified version of a magnetic bead-based radioimmunoassay(Advanced Magnetics, Cambridge, Mass.). The final antigen/antibodycomplex was separated from free ¹²⁵I-cAMP by vacuum filtration through aPVDF filter in a microliter plate (Millipore, Bedford, Mass.). Filterswere punched and counted for ¹²⁵I in a Packard gamma counter. Bindingdata were analyzed using nonlinear regression and statistical techniquesavailable in the GraphPAD® Prism package (San Diego, Calif.).

[0168] Functional Assay: Intracellular Calcium Mobilization

[0169] The intracellular free calcium concentration was measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM.Stably transfected cells were seeded onto a 35 mm culture dishcontaining a glass coverslip insert. Cells were washed with HBS and thenloaded with 100 μl of Fura-2/AM (10 μM) for 20 to 40 min. After washingwith HBS to remove the Fura-2/AM solution, cells were equilibrated inHBS for 10 to 20 min. Cells were then visualized under the 40× objectiveof a Leitz Fluovert FS microscope and fluorescence emission wasdetermined at 510 nM with excitation wave lengths alternating between340 nM and 380 nM. Raw fluorescence data were converted to calciumconcentrations using standard calcium concentration curves and softwareanalysis techniques.

[0170] Reagents

[0171] Cell culture media and supplements were from Specialty Media(Lavallette, N.J.). Cell culture plates (150 mm) were from Corning(Corning, N.Y.). Cell culture flasks (225 cm²) and polypropylenemicroliter plates were from Co-star (Cambridge, Mass.). Porcine ¹²⁵I-PYYwas from New England Nuclear (Boston, Mass.). NPY and related peptideanalogs were from either Bachem Calif. (Torrance, Calif.) or Peninsula(Belmont, Calif.). Whatman® GF/C filters were Brandel (Gaithersburg,Md.). Bio-Rad Reagent was from Bio-Rad (Hercules, Calif.). Bovine serumalbumin and bacitracin were from Sigma (St. Louis. Mo.). All othermaterials were reagent grade.

[0172] Results

[0173] Isolation of a cDNA Clone Encoding a Human Hippocampal Y2Receptor

[0174] In order to clone a human NPY receptor subtype (Y2), we used anexpression cloning strategy in COS-7 cells (20, 21, 22). This strategywas chosen for its extreme sensitivity since it allows detection of asingle “receptor positive” cell by direct microscopic autoradiography.

[0175] Since the Y2 receptor is described as a presynaptic receptor, itis difficult to locate cell bodies that actually contain this specificmRNA in restricted brain areas. We reasoned that human hippocampus was agood source of mRNA since it contains both a large number ofinterneurons and has been shown to carry a particularly dense populationof Y2 receptors (23, 24, 25, 26). A human hippocampal cDNA library of2.2×10⁶ independent recombinants with a 3 kb average insert size wasfractionated into 440 pools of ≈5000 independent clones. From the first200 pools tested, three gave rise to positive cells in the screeningassay (#145, 158 and 189). The last 220 pools tested were all negative.

[0176] Since both Y1 and Y2 receptor subtypes are expressed in thehippocampus (2), we analyzed the DNA of positive pools by PCR with Y1specific primers. Pools #145 and #158 turned out to contain cDNAsencoding an Y1 receptor, but pool #189, negative by PCR (data notshown), likely contained a cDNA encoding a human hippocampal NPYreceptor that was not Y1. Pool #189 was subdivided in 20 pools of 1000clones each, and a preliminary pharmacological characterization was runon COS-7 cells transfected with DNA prepared from the secondary pools.This preliminary analysis revealed that a 100 fold excess of cold[Leu³¹-Pro³⁴]NPY totally inhibited binding of ¹²⁵I-PYY to control COS-7cells transfected with the Y1 gene. In contrast, no significantinhibition of binding was observed when the same experiment wasperformed on COS-7 cells transfected with secondary pool #189-17 (datanot shown). This is consistent with pool #189 containing a cDNA encodinga human hippocampal Y2 receptor. The sib selection was therefore pursuedon pool #189 until a single clone was isolated (designated CG-13).

[0177] The isolated clone carries a 4.2 kb cDNA. This cDNA contains anopen reading frame between nucleotides 1002 and 2147 that encodes a 381amino acid protein (SEQ. I.D. No. 2). The unusually long 5′ untranslatedregion could be involved in the regulation of translation efficiency ormRNA stability. The flanking sequence around the putative initiationcodon conforms to the Kozak consensus sequence for optimal translationinitiation (27, 28).

[0178] The hydrophobicity plot displayed seven hydrophobic, putativemembrane spanning regions which makes the human hippocampal Y2 receptora member of the G-protein coupled superfamily. The nucleotide anddeduced amino acid sequences are shown in FIG. 1 and FIG. 2,respectively.

[0179] Like most G-protein coupled receptors, the Y2 receptor contains aconsensus sequence for N-linked glycosylation, in the amino terminus(position 11) involved in the proper expression of membrane proteins(29). The Y2 receptor carries two highly conserved cysteine residues inthe first two extracellular loops that are believed to form a disulfidebond stabilizing the functional protein structure (30). The Y2 receptorshows 7 potential phosphorylation sites for protein kinase C inpositions 11, 27, 64, 145, 188, 250 and 340, 2 casein kinase sites inpositions 174 and 358, and 2 cAMP- and cGMP-dependent protein kinasephosphorylation sites in positions 146 and 350. It should be noted that7 of those 11 potential phosphorylation sites are located inintra-cellular loops 1, 2 and 3 as well as in the carboxyl terminus ofthe receptor and therefore could play a role in regulating functionalcharacteristics of the Y2 receptor (30). A potential palmitoylation siteis present in the sequence at the cysteine found in position 326. Alarge number of G-protein coupled receptors carry a cysteine in the sameposition and O'Dowd et al. have speculated that it plays an importantrole in the functional coupling of the human β₂-adrenergic receptor(31). The formation of this additional cytosolic loop may influence themobility of the receptor across the membrane (32).

[0180] When compared to the published human Y1 cDNA clone (10, 11) theY2 sequence shows surprisingly low homology both at the nucleotidelevel, 48.1% (FIG. 3) and overall amino acid level, 31% (FIG. 4). Thetransmembrane domain identity of the human hippocampal Y2 receptor withother 7 TM receptors is shown in Table 1. The low TM identity with otherG-protein coupled receptor families, with other peptide receptors andespecially with the Yl subtype raises the possibility that Y2 receptorsubtypes belong to a new distinct sub-family of 7 TM peptide receptors.Conversely, NPY receptor subtypes could form a sub-family where membersshow unusually low levels of overall homology. Applicants have alsocloned the human Y4 receptor, and this receptor also exhibits a lowdegree of homology with the human Y2 receptor (Table 1). It isinteresting to observe that the mouse orphan receptor MUSGIR (mouseglucocorticoid induced receptor, 33) shows the highest TM identity (42%,Table 1) with our human Y2 receptor. The same comparison between humanY1 (or Y4) and Y2 TM regions only gives a score of 41% identity. If wewere comparing the human Y2 receptor sequence with the human homolog ofthe MUSGIR receptor, the level of identity might even be higher.Therefore the MUSGIR receptor could be related to the NPY receptors andbind members of the pancreatic polypeptide ligand family. A fullpharmacological evaluation of the human GIR homolog with NPY, PYY and PPrelated ligands is now underway to verify this hypothesis.

[0181] Using the human Y2 probe, northern hybridizations reveal a uniqueband at 4.3 kb in human brain after a three-day exposure (FIG. 16). Thisis in good agreement with the 4.2 kb cDNA that we isolated by expressioncloning and indicates that our cDNA clone is full-length. The mRNAencoding the human Y2 receptor is present in significant amounts inamygdala, corpus callosum, hippocampus, and subthalamic nucleus. A faintband is detectable in caudate nucleus, hypothalamus and substantianigra. No signal could be detected in thalamus. It should be noted thatClontech's MTN blot does not carry any mRNA from cortex or brain stem.

[0182] Southern hybridizations to human genomic DNA followed by highstringency washes (FIG. 17) suggest that the human genome contains asingle Y2 receptor gene (single band with EcoRI, HindIII, BamHI andPstI). The faint bands at 9 and 12 kb observed with BglII can beexplained by the presence of two BglII restriction sites in the codingregion of the human Y2 sequence and are also consistent with a single Y2receptor gene. Pharmacology of the transiently expressed human Y2receptor The Y2-like pharmacology of CG-13, originally identified bywhole cell autoradiographic techniques, was further defined by membranebinding assays. The gene for the human hippocampal Y2 receptor wastransiently expressed in COS-7 cells for full pharmacologicalevaluation. ¹²⁵I-PYY bound specifically to membranes from COS-7 cellstransiently transfected with the CG-13 construct. The time course ofspecific binding was measured in the presence of 0.06 nM ¹²⁵I-PYY (FIG.5). The association curve was biphasic, with approximately 55% of thespecific binding occurring during an initial rapid phase and 45%following a slower time course. For the rapid phase, the observedassociation constant (K_(obs)) was 1.28±0.02 min⁻¹ and t_(1/2) was 0.5min; equilibrium binding was 95% complete within 2 min and 100% completewithin 5 min (n=3). For the slow phase, K_(obs) was 0.02±0.00 min⁻¹ andt_(1/2) was 37 min; equilibrium binding was 90% complete within 120 min,95% complete within 160 min and 100% complete within 280 min (n=3).Total equilibrium binding, composed of both phases, was 95% completewithin 120 min and 100% complete within 240 min. The biphasicassociation curve may reflect a complex pattern of receptor surfacebinding followed by access to deep-seated binding sites, as has beensuggested by Schwartz and co-workers for Y2 receptors (34). Forcomparison, we also measured the time course of binding to human Y1receptors transiently expressed in COS-7 cells (FIG. 5). The associationcurve was monophasic, with a K_(obs) of 0.06±0.02 min⁻¹ and a t_(1/2) of12 min; equilibrium binding was 95% complete within 51 min and 100%complete within 90 min (n=3). The different patterns of association forCG-13 and human Y1 receptors suggest novel mechanisms of receptor/ligandinteraction.

[0183] Saturation binding data for ¹²⁵I-PYY were fit to a one-site modelwith an apparent K_(d) of 0.069±0.009 nM and an apparent B_(max) of7.8±0.4 pmol/mg membrane protein, corresponding to approximately 7.5×10⁵receptors/cell (n=3; FIG. 6). Given that the transfection efficiency was20-30% (data not shown), the receptor density on transfected cells wasprobably closer to 3×10⁶/cell. Membranes from mock-transfected cells,when prepared and analyzed in the same way as those fromCG-13-transfected cells, displayed no specific binding of ¹²⁵I-PYY. Weconclude that the ¹²⁵I-PYY binding sites observed under the describedconditions were derived from the CG-13 construct.

[0184] Y2 receptor recognition is thought to depend primarily upon thefour C-terminal residues of NPY (Arg³³-Gln³⁴-Arg³⁵-Tyr³⁶-NH₂) precededby an amphipathic α-helix (M4, M5); exchange of Gln³⁴ with Pro³⁴ is notwell tolerated (4, 5). We therefore chose several C-terminal fragmentsand C-terminal modified peptides for competition binding studies. Therank order of affinity for selected compounds was derived fromcompetitive displacement of ¹²⁵I-PYY (FIG. 7 and Table 3). The CG-13receptor was compared with two model systems: 1) the cloned human Y1receptor (10, 11) transiently expressed in COS-7 cells, and 2) theY2-like receptor population expressed by human SK—N—Be(2) neuroblastomacells (2, 8). To our knowledge, no models for human Y3 and human PPreceptors have been described.

[0185] CG-13 bound with high affinity to human NPY (K_(i)=0.69 nM) andeven more so to human PYY (K_(i)=0.39 nM). The K_(i) values are inagreement with numerous reports of pharmacologically defined Y2receptors studied in NPY binding and functional assays (2). The oppositerank order was observed with human Y1 receptors, combined with strongerreceptor/binding interactions (K_(i)=0.049 and 0.085 nM for human NPYand human PYY, respectively). It is interesting in this regard thatCG-13 bound ¹²⁵I-PYY (K_(d)=0.069 nM) with higher affinity than PYY(K_(i)=0.39 nM), suggesting that iodination may stabilize thereceptor/ligand complex. The human Y1 receptor, in contrast, bound both¹²⁵I-PYY (K_(d)=0.062+0.010 nM, n=3, data not shown) and PYY(K_(i)=0.085 nM) with comparable affinity. The fact that CG-13 and thehuman Y1 receptor bound NPY, PYY and ¹²⁵I-PYY with different magnitudesand rank orders of affinity most likely reflects distinct mechanisms ofpeptide recognition which could potentially be exploited for thedevelopment of subtype-selective non-peptide ligands.

[0186] CG-13 also bound with high affinity to porcine NPY (K_(i)=0.86nM), which differs from human NPY by containing Leu¹⁷ in the PP-foldrather than Met¹⁷. CG-13 was relatively insensitive to N-terminaldeletion of NPY and PYY; the affinity for porcine NPY₂₂₋₃₆ was only5-fold less than that for full length porcine NPY. Extreme deletion ofα-helical structure was less well tolerated; the affinity for porcineNPY₂₆₋₃₆ was 240-fold less than that for full length porcine NPY. Human[Leu³¹,Pro³⁴]NPY and human PP, both having Pro³⁴ rather than Glu³⁴, didnot bind well (K_(i)>300 nM). Hydrolysis of the carboxyl terminal amideto free carboxylic acid, as in NPY free acid, also disrupted bindingaffinity for CG-13 (K_(i)>300 nM). The terminal amide appears to be acommon structural requirement for pancreatic polypeptide family/receptorinteractions.

[0187] The competitive displacement data indicate that CG-13 binds PYYwith equal or greater affinity than NPY. The C-terminal region of NPY isthe primary pharmacophore. CG-13 does not tolerate exchange of Gln³⁴with Pro³⁴, as revealed by low affinity interactions with human[Leu³¹,Pro³⁴]NPY and human PP. The binding profile, which is shared bySK—N—Be(2) cell receptors but not by human Y1 receptors, ischaracteristic of the pharmacologically defined Y2 receptor (refs. 2, 8;see also Table 2). The membrane binding studies therefore confirm andextend our assessment that CG-13 encodes a human Y2 receptor.

[0188] The pharmacological profile of the human Y2 receptor was furtherinvestigated using peptide analogs related to NPY, PYY, and PP (Table4). CG-13 did not discriminate human and frog analogs of NPY (K_(i)=0.74and 0.87 nM, respectively), human and porcine analogs of NPY₂₋₃₆(K_(i)=2.0 and 1.2 nM, respectively), human and porcine analogs of[Leu³¹, Pro³⁴]NPY (K_(i)>130 and >540 nM, respectively), or human NPYand human [Tyr-O-Me²¹]NPY (K_(i)=0.74 and 1.6 nM, respectively). Thislast derivative was tested based on the proposal that it was selectivefor central vs. peripheral NPY receptors, with high binding affinity inrat CNS but low potency in rat vas deferens relative to NPY (83). Forthe receptors under investigation, however, [Tyr-O-Me²¹]NPY and humanNPY yielded highly similar binding profiles. The NPY derivative withgreatest selectivity for CG-13 was C2-NPY, a C² to C²⁷disulfide-stabilized derivative of NPY with an 8-amino-octanoic linkerreplacing NPY₅₂₄ (K_(i)=3.5 nM, >20-fold selective for CG-13 over Y1 andY4 receptors). C2-NPY has been described as a Y2-selective compound (3).

[0189] Three additional PYY derivatives yielded distinctive bindingprofiles. CG-13 bound with highest affinity and greatest selectivity tohuman PYY₃₋₃₆ (K_(i)=0.70 nM, >20-fold selective for CG-13 over Y1 andY4 receptors). PYY₃₋₃₆ is a major form of PYY-like immunoreactivity inblood and could therefore mediate CG-13-dependent processes in vivo (84,85). Porcine PYY was relatively nonselective and similar in bindingaffinity to human PYY (K_(i)=0.35 nM and 0.36 nM, respectively). Human[Pro³⁴]PYY was lacking in binding affinity for CG-13 (K_(i)>310),further supporting the argument that Pros is disruptive for highaffinity peptide binding to the CG-13 receptor.

[0190] Six additional PP derivatives were investigated. Those peptideswhich resemble human PP in that they contain Pro³⁴ (bovine, rat, avian,and frog PP) displayed no activity in the CG-13 binding assay. Highaffinity binding was detected only for salmon PP (K_(i)=0.1 nM), whichis distinguished by containing Gln³⁴. When the C-terminus of human PPwas modified to more closely resemble human NPY, as in [Ile³¹, Gln³⁴]PP,the binding affinity for CG-13 was increased dramatically (K_(i)=20 nM).It has been reported previously that [Ile³¹, Gln³⁴]PP was more activethan PP in Y2 binding assays, while exhibiting decreased potency forputative PP receptors in rat vas deferens (86).

[0191] Several proposed NPY antagonists were analyzed for their abilityto bind to CG-13 receptors. These include PYX-1 and PYX-2, C-terminalderivatives of NPY reported to antagonize NPY-mediated feeding andneurotransmitter release (87, 88, 89). Neither synthetic peptide boundto CG-13 with high affinity or selectivity (K_(i)=684 for PYX-1 andK_(i)>1000 nM for PYX-2). [D-Trp³²]NPY is an NPY derivative reported toregulate feeding behavior when injected into the hypothalamus of rats(90); this analog was inactive in the CG-13 binding assay. Anotherinactive compound was NPY₁₂₄ amide, a peptide reported to antagonize NPYin the rat vas deferens (83).

[0192] Human Tissue Y2 Receptor Macrolocalization: PCR

[0193] Human Y2 mRNA was detected by PCR techniques in a broad range ofhuman tissues (Table 5). Relatively intense hybridization signals weredetected in total brain, thoracic artery, coronary artery, and penis,with more moderate levels in frontal brain, ventricle, mesentery,stomach and ileum. Relatively low levels were detected in nasal mucosaand pancreas. Several other tissues were negative for Y2 mRNA asmeasured by this technique, including atrium, liver, and uterus.

[0194] Cloning and Expression of Two Isoforms of the Rat NPY/PYY (Y2)Receptor

[0195] Two rat genomic clones homologous to the human Y2 receptor wereisolated, termed rs5a (FIG. 8) and rs26a (FIG. 9). The nucleotidesequence of rs5a is 86.5% identical in the coding region to that of thehuman Y2 receptor (FIG. 10), and can encode a 381 amino acid proteinwith 94.5% identity to the human Y2 amino acid sequence (FIG. 11). Inthe putative transmembrane domains (TMs), the protein predicted by rs5aexhibits 98.2% amino acid identity with the human Y2 receptor (FIG. 11).This high degree of primary sequence identity is often observed forspecies homologues, and strongly suggests that the receptor encoded byrs5a is the rat Y2 receptor. However, even a single amino acidsubstitution can influence the functional properties of a receptor;thus, even species homologues exhibiting a high level of sequenceidentity may display different pharmacological properties (infra),underscoring the importance of obtaining both rat and human receptorsfor use in drug development.

[0196] Sequence analysis of the second genomic clone revealed that rs26aalso encoded a full-length rat Y2 receptor; however, rs26a contains twonucleotide changes when compared with the sequence of rs5a. Bothnucleotide changes result in amino acid substitutions in the predictedrat Y2 receptor protein. With two (2) amino acid changes, the proteinencoded by rs26a is 99.7% identical to that of rs5a. Compared with thehuman Y2 receptor, the nucleotide sequence identity of rs26a is 85.2%and the amino acid sequence identity is 98.2%. This clone thereforeencodes an isoform of the rat Y2 receptor distinct from that encoded byrs5a. The locations of the amino acid substitutions (N-terminus and ⅚loop; see FIG. 3) suggest that they could potentially influence receptorfunction. The Y2 receptors encoded by rs5a and rs26a are likely torepresent allelic variants at the same gene locus; however, rs26a couldrepresent a second rat Y2 gene. Accordingly, we have designated theisoform encoded by rs5a as the rat Y2a receptor, and designated theisoform encoded by rs26a as the rat Y2b receptor.

[0197] The primary sequences of rat and human Y2 receptors, while highlyrelated, show distinct patterns of sequence motifs for N-linkedglycosylation, N-myristoylation, and protein phosphorylation. Forexample, the rat Y2a differs from the rat Y2b in that it contains anadditional site for phosphorylation by protein kinase C. Further, thehuman Y2 differs from both rat Y2 isoforms in containing two additionalsites for N-linked glycosylation, two additional sites for cAMP- andcGMP-dependent protein phosphorylation, an additional site for caseinkinase II phosphorylation, one additional site for protein kinase Cphosphorylation, and two fewer sites for N-myristoylation. These sitescould mediate differences in the function or regulation of the threereceptors. The isolation of two rat homologues of the Y2 receptorprovides the means to compare the pharmacological properties of the ratand human Y2 receptors (see below) in relation to their observeddifferences in primary structures. These data will be critical to thedesign and testing of human therapeutic agents acting at these sites.

[0198] Binding Studies with Rat Y2 Homologs

[0199] The DNA corresponding to the rat Y2a homolog was transientlyexpressed in COS-7 cells for membrane binding studies. The binding of¹²⁵I-PYY to the rat Y2a receptor was saturable over a radioligandconcentration of 0.5 pM to 2.5 nM. Binding data were fit to a one-sitemodel with an apparent K_(d)=0.26 nM and a receptor density of 5100fmol/mg membrane protein. As determined by using peptide analogs withinthe pancreatic polypeptide family, the rat Y2a pharmacological profileresembles that for the human Y2 receptor (Table 6). Each receptor analogis relatively tolerant of N-terminal ligand deletion (the humanapparently more so than the rat) and intolerant of any peptidecontaining Pros or a modified C-terminus (as in NPY free acid or[D-Trp³²]NPY).

[0200] The rat Y2b clone, which differs from rat Y2a by two amino acidchanges one in the N-terminal tail (from Leu²⁰ to Phe²⁰) and another inthe third intracellular loop (from Thr²⁶⁶ to Met²⁶⁶), has been subjectedonly to a preliminary investigation. Membranes from COS-7 cellstransiently transfected with the rat Y2b receptor were incubated with0.08 nM ¹²⁵I-PYY and analyzed for specific binding after incubation at30° C. for 120 min. Membranes from transfected cells bound 310 fmol¹²⁵I-PYY/mg membrane protein, whereas membranes from mock-transfectedcells (receiving vector without receptor cDNA insert) bound only 3 fmol¹²⁵I-PYY/mg membrane protein. It remains to be determined whether thereexist any pharmacological or functional differences between the ratY2aand rat Y2b receptors.

[0201] Localization of NPY Y2 Messenger RNA in the Rat Central NervousSystem

[0202] In control experiments, hybridization signals for rat NPY Y2 mRNAwere seen only with the antisense probes (probe sequences shown in Table7), and only over cells which had been transfected with the rat Y2 DNA(FIG. 18). The probes were designed to recognize both rat Y2a and ratY2b. Neither mock transfected cells nor cells transfected with rat NPYY1 mRNA exhibited hybridization signals. On rat brain sections, nohybridization signals were obtained with the sense probes, only with theantisense probes.

[0203] The distribution of NPY Y2 mRNA observed in coronal sectionsthrough the rostrocaudal extent of rat brain is shown in FIG. 12 andTable 8. Hybridization signals were seen over many areas of the ratbrain (FIG. 12), which, at the microscopic level, were confined to thecytoplasm of neuronal profiles (data not shown). In the telencephalon,the most intense hybridization signals were observed over the CA3 regionof the hippocampus (FIGS. 12B-E) and over the anteroventral aspect ofthe medial nucleus of the amygdala (FIGS. 12C, D). Less intense signalswere found over the olfactory tubercle, the lateral septal nucleus (FIG.12A), and over the basomedial nucleus and posteromedial cortical nucleusof the amygdala (FIGS. 12D, E). Scattered neurons with hybridizationsignal were also seen in the central amygdaloid nucleus. In cortex,silver grains were seen over large neurons in the piriform region. Amongdiencephalic structures, the arcuate nucleus of the hypothalamusexhibited the most intense hybridization signal for NPY Y2 mRNA (FIGS.12D, E). In this area, most of the neurons appeared to be labelled, andmany neurons were also labelled in the region of the tuber cinereumlateral to the arcuate nucleus. In addition, both the dorsomedial andventromedial hypothalamic nuclei contained appreciable hybridizationsignals over subpopulations of neurons (FIGS. 12C, D). In the dorsal andventral premammillary nuclei, hybridization signal was seen over manyneurons (FIG. 12E). In the thalamus, neurons in the centromedial nucleuswere labelled (FIGS. 12C, D), while a smaller, less intensely labelledgroup of cells was visible in the paraventricular nucleus (FIG. 12D).

[0204] In the mesencephalon, medulla, and pons, few structures werelabelled with the antisense oligonucleotide probe. Those exhibiting amoderate level of hybridization signal were the dorsal and caudal linearraphe (FIG. 12F), the pontine nucleus, and the posterior dorsaltegmental nucleus (FIG. 12G). In the spinal cord, labelling was observedover scattered large neurons in lamina 9 (FIG. 12H). Silver grains werealso found over a few large neurons in the dorsal root ganglion.

[0205] Receptor/G Protein Interactions: Effects of Guanine Nucleotides

[0206] For a given G protein-coupled receptor, a portion of the receptorpopulation in a membrane homogenate typically exists in the highaffinity ligand binding state as a receptor/G protein complex. Thebinding of GTP or a non-hydrolyzable analog to the G protein causes aconformational change in the receptor which favors a low affinityligand/binding state (110). We investigated whether the non-hydrolyzableGTP analog, Gpp(NH)p, would alter the binding of human NPY or ¹²⁵I-PYYto Y2 receptors transiently expressed in COS-7 cells. The competitioncurve produced by human NPY was evaluated in the absence and presence of100 μM Gpp(NH)p. The human Y2 receptor was relatively insensitive to theGpp(NH)p compared to the rat Y2a receptor (FIG. 13). The IC₅₀ for humanNPY binding to the human Y2 receptor was increased from 2.2 nM to 3.3nM; specific binding of 125I-PYY was decreased by only 4% (n=5). TheIC₅₀ for human NPY binding to the rat Y2a receptor was altered verylittle (from 0.7 nM to 1.2 nM, n=2); specific binding of ¹²⁵I-PYY,however, was decreased by 23% (n=2). A similar pattern of sensitivity toGpp(NH)p was reported for ¹²⁵I-PYY binding to rat brain (91). Thedifference between the rat and human Y2 receptor clones could beexplained by several factors, including 1) the types of G proteinsavailable in COS-7 cells, 2) the level of receptor reserve in COS-7cells (note that human Y2 receptor density was greater than that of therat Y2a receptor), and 3) the efficiency of receptor/G protein coupling(92; 93).

[0207] Stable Expression Systems: Characterization in Binding Assays

[0208] Untransfected 293 and NIH-3T3 cells were pre-screened forspecific ¹²⁵I-PYY binding and found to be negative (data not shown).After co-transfection with the human Y2 cDNA plus a G-418-resistant geneand selection with G-418, surviving colonies were screened for specificbinding of ¹²⁵I-PYY. Two positive clones were identified and isolatedfor further study (293 clone #10 and NIH-3T3 clone #5). The binding of¹²⁵I-PYY to membranes from the 293 stable clone was saturable over aradioligand concentration range of 0.5 pM to 2.5 nM. Binding data werefit to a one-site binding model with an apparent K_(d) of 3±1 pM and areceptor density of 880±50 fmol/mg membrane protein (mean±s.e.m., n=3).Membranes from stably transfected NIH-3T3 cells displayed similarbinding properties, with an apparent K_(d) of 8±2 pM and a receptordensity of 160±60 fmol/mg membrane protein (mean±s.e.m., n=2). Membranesfrom both stable clones were incubated with 0.08 nM ¹²⁵I-PYY in thepresence or absence of 100 μM Gpp(NH)p. Specific binding of ¹²⁵I-PYY toY2 receptors in 293 cell membranes was reduced 32% in the presence ofthe guanine nucleotide, whereas specific binding to Y2 receptors inNIH-3T3 cell membranes was reduced only 6% under the same conditions.The data serve to emphasize that the receptor/G protein interactions fora given receptor clone can vary depending upon the resident G proteinsin the host cell line (93). Additional factors such as receptor densityand receptor reserve can also play a role (92).

[0209] Functional Assay: cAMP

[0210] Activation of all Y-type receptors described thus far is thoughtto involve coupling to G-proteins which are inhibitory for adenylatecyclase activity (G_(i) or G_(o)) (1). Based on these priorobservations, we investigated the ability of PYY to inhibitforskolin-stimulated cAMP accumulation in 293 cells stably expressingthe human Y2 receptor. Incubation of intact cells with 10 μM forskolinproduced a 10-fold increase in cAMP accumulation over a 5 minute period,as determined by radioimmunoassay. Simultaneous incubation with humanPYY decreased the forskolin-stimulated cAMP accumulation by 71% instably transfected 293 cells (FIG. 14) but not in untransfected cells(data not shown). The NPY-mediated response was concentration-dependent(EC₅₀=0.25 nM). We conclude that human Y2 receptor activation can resultin decreased cAMP accumulation, very likely through inhibition ofadenylate cyclase activity. Similar results were obtained for NIH-3T3cells stably transfected with the human Y2 receptor, in which human NPYdecreased forskolin-stimulated cAMP accumulation by 50% in transfectedcells with an EC₅₀ of 0.21 nM (FIG. 14).

[0211] Peptides selected for their ability to bind to the transientlyexpressed human Y2 receptor were further investigated for functionalactivity using stably transfected 293 cells (Table 9). All peptides withmeasurable binding affinity were able to mimic the effects of PYY oncAMP accumulation. EC₅₀ values were generally within a 10-fold range ofK_(i) values, often lower in magnitude (Table 9). We also investigatedthe functional activity of the reported feeding behavior modulator[D-Trp³²]NPY. Consistent with this peptide's low binding affinity forthe human Y2 receptor, we detected no functional activity atconcentrations up to 0.3 μM, or when tested at 0.3 MM for antagonism ofthe functional response (data not shown). The reported NPY receptorantagonists PYX-1 and PYX-2 were also inactive when tested under thesame paradigm.

[0212] Functional Assay: Intracellular Calcium Mobilization

[0213] The intracellular free calcium concentration was increased in 293cells stably transfected with the human Y2 receptor after application of1 μM human PYY (Δ [Ca²⁺]_(i)=80 nM; FIG. 15). The PYY-mediated responsewas concentration-dependent, with EC₅₀=39 nM, n=2 (FIG. 15). PYY-inducedcalcium mobilization was relatively maintained in the presence of 1 mMextracellular EGTA (Δ [Ca²⁺]_(i)=64 nM for 1 μM human PYY), suggestingthat intracellular calcium stores are the primary source of thetransient calcium flux. Pretreatment with pertussis toxin (100 ng/ml for24 hours) decreased the response to 300 nM human PYY by 93%, therebysupporting a G protein-linked signal transduction pathway. Untransfected293 cells did not respond to human PYY (data not shown). The calciummobilization assay provides a second pathway through which Y2 receptoractivation can be measured.

[0214] Discussion

[0215] Attempts to isolate the NPY Y2 receptor subtype based on sequencehomology with the Y1 receptor have not been successful so far.Therefore, we chose an expression cloning approach where a functionalreceptor is actually detected with exquisite sensitivity on the surfaceof transfected cells, using a highly specific iodinated ligand. Usingthis strategy, we have identified a human cDNA encoding thepharmacologically defined Y2 receptor. The fact that we had to screen2.2×10⁶ independent clones with a 3 kb average insert size to find oneclone reveals either a very strong bias against Y2 cDNA cloning in thecDNA library construction procedure, or the Y2 mRNA is expressed at verylow levels in human hippocampal tissue. The longest reading frame in thecDNA encodes a 381 amino acid protein with an estimated molecular weightof 42 kD. Given the fact that there is an N-linked glycosylation site inthe amino terminus, the apparent molecular weight could be slightlyhigher and in good agreement with published data on the molecular weightof the human hippocampal Y2 receptor at 50 kD (36). The Y2 receptorcarries a large number of potential phosphorylation sites which could beinvolved in the regulation of its functional characteristics.

[0216] The nucleotide and amino acid sequence analysis both reveal lowidentity levels with all 7-TM receptors including the human Y1 and Y4receptors. The highest transmembrane amino acid identity is found withthe mouse MUSGIR receptor. A pharmacological profile on the human GIRhomolog will be established with NPY, PYY and pancreatic polypeptiderelated ligands to find out if this orphan receptor belongs to the samepharmacologically defined neuropeptide Y receptor sub-family. The humanY2 receptor shares very low amino acid identity with the previouslycloned human Y1 receptor (31% overall and 41% in transmembrane regions).The human Y2 receptor also displays a unique pharmacological profile anda unique time course of association with ¹²⁵I-PYY. The dramaticdifferences in sequence and pharmacological profile between the human Y1and Y2 receptors suggest that they might be encoded by two unrelatedgenes whose products have evolved into binding the same family ofligands. Conversely, they could have diverged from a common ancestorvery early in evolution and undergone multiple mutations leading todistinct pharmacological characteristics.

[0217] Northern analysis reveals a 4.3 kb band in human brain anddemonstrates that our 4.2 kb Y2 cDNA is full-length. Southern analysesare consistent with the human genome containing a single Y2 receptorgene.

[0218] The pharmacological binding profile established in our initialcharacterization served primarily to establish the CG-13 as a human Y2receptor. The additional data included here reflect an increasedunderstanding of receptor ligand/interactions. We now know, for example,that C2-NPY and PYY₃₋₃₆ can be used to compete for Y2 receptor siteswith greater affinity and selectivity than the C-terminal fragments ofNPY originally described. We also know that certain peptides which arethought to antagonize NPY-dependent effects, such as [D-Trp³²]NPY,PYX-1, and PYX-2, are unable to compete for binding of the human Y2receptor clone described here. Our evidence does not therefore supportthe cloned Y2 receptor as the molecular target of these particularpeptides in vivo or in vitro.

[0219] Human Y2 receptor mRNA was detected by PCR techniques in a broadrange of human tissues (Table 5). Relatively intense hybridizationsignals were detected in total brain, thoracic artery, coronary artery,and penis, with more moderate levels in frontal brain, ventricle, andmesentery. This distribution is consistent with evidence for Y2 receptorlocalization and receptor-dependent effects in CNS, cardiovascular, andreproductive physiology (94). Moderate hybridization signals were alsodetected in stomach and ileum, consistent with evidence for Y2-mediatedeffects on chief cell cAMP accumulation (95) and also intestinalelectrolyte flux (61; 96). Relatively low levels were detected in nasalmucosa and pancreas, two tissues in which Y2-like receptors have beenreported to regulate vasoconstriction and pancreatic secretion,respectively (97, 98, 99). A more definitive localization of the Y2receptor mRNA and receptor expression (i.e., whether on neurons,enterocytes, vascular smooth muscle cells, etc.) is attainable throughin situ hybridization and receptor autoradiography techniques.

[0220] The distribution of NPY Y2 mRNA described here in rat brain has anumber of potential implications, and raises a number of importantquestions. Among these are; 1) how does the distribution of this mRNAcorrelate with that of NPY itself; 2) how does the Y2 mRNA distributionrelate to the putative autoradiographic localization of Y2 receptorsdescribed by previous investigators; and 3) what are the functionalimplications of the Y2 mRNA distribution?

[0221] Correlation with NPY Immunoreactivity

[0222] Neuropeptide Y is one of the most abundant and widely distributedpeptides in the mammalian brain (100). In some areas, NPY Y2 mRNAappears to be co-distributed with NPY-immunoreactive (NPYir) neurons,although colocalization in the same neuron(s) remains to be established.In both the arcuate nucleus of the hypothalamus and the medial nucleusof the amygdala, the distribution of Y2 mRNA overlaps with thedistribution of NPYir neurons demonstrated by immunocytochemical studies(100, 101). In addition, both areas contain moderate plexuses of NPYiraxons. These observations leave open the question ofpresynaptic/postsynaptic nature of the Y2 receptor. In most other areasof the brain, the Y2 mRNA does not appear to be co-distributed withNPYir neurons, but instead correlates better with the distribution ofNPYir terminal fields, suggesting a postsynaptic localization.

[0223] Comparison with Receptor Autoradiography

[0224] A number of investigators have described the distribution of NPYreceptors based on the autoradiographic localization of radiolabelledNPY ligands, among them [¹²⁵I]NPY and [¹²⁵I]peptide YY (PYY), incombination with subtype-selective displacers. The Y2 receptor has beenlocalized by combining (¹²⁵I)PYY with the Y2-selective mask NPY₁₃₋₃₆(94). The results of such studies suggest that the Y2 receptor is widelydistributed in rat brain, being most abundant in the hippocampus,olfactory bulb, and hypothalamus. We have seen no NPY Y2 mRNA in theolfactory bulb, but both hippocampus and hypothalamus contain Y2 mRNA.However, the pharmacological characterization of NPY receptor subtypesis incomplete at present, and some of the Y2-like binding may beattributable to the so-called a typical Y1 receptor, or to otherundiscovered NPY receptor subtypes.

[0225] Our in situ results suggest that the receptor autoradiographiccharacterization of the Y2 receptor is likely to be accurate for someareas. The projection fields of neurons containing the Y2 mRNA areimportant in this respect. Thus the pyramidal neurons of the CA3 regionof the hippocampus, which contain relatively intense Y2 hybridizationsignals, project in a topographic fashion to the lateral septum (102),an area which supposedly contains a high proportion of Y2 receptors(103, 23, 94). Similarly, the olfactory bulb appears to contain mainlyNPY receptors of the Y2 subtype. While there is no Y2 mRNA in theolfactory bulb, the piriform cortex contains many neurons which arelabelled with the Y2 antisense probe, and provides a major source ofolfactory bulb afferents. The localization of NPY Y2 mRNA in the arcuatenucleus of the hypothalamus is particularly interesting, as NPYirneurons in this nucleus provide the NPY innervation of much of thehypothalamus, including the paraventricular and dorsomedial nuclei (104,105). It is unclear at present which receptor subtype(s) predominate inthe paraventricular nucleus, but based on our results with the Y2 mRNA,and those of Mikkelsen and colleagues with the Y1 mRNA (106, 107), bothY1 and Y2 should be present. Similar arguments can be pursued for mostof the regions which contain Y2 mRNA, however a definitive profile of Y2receptor localization awaits the introduction of Y2 selective ligands.

[0226] Functional Considerations

[0227] Neuropeptide Y is involved in a number of physiologicalfunctions, including the regulation of food intake, neuronalexcitability, cardiovascular regulation, and circadian rhythms. Withregard to food intake, the paraventricular nucleus of the hypothalamusis one site which has been intensively investigated, and has beendemonstrated to be a prominent locus of action for the orexigeniceffects of NPY. The localization of NPY Y2 mRNA in the arcuate nucleus,and the projections of the arcuate to the paraventricular nucleus,suggest the involvement of this receptor in feeding.

[0228] In the hippocampus, NPY immunoreactivity is found mainly ininterneurons which innervate pyramidal cells. Here, NPY has beendemonstrated to reduce synaptic excitation in areas CA1 and CA3. Thishas been assumed to be mediated by a Y2 receptor (108), as C-terminalfragments of NPY are effective in the assay. The localization of Y2 mRNAin pyramidal cells of CA3 indicates that this receptor may be involvedin the termination of convulsive activity, such as in epilepsy.

[0229] The rat Y2a and Y2b receptor analogs represent essential toolsfor pharmaceutical drug development. Drug candidates screened primarilyagainst human receptors must also be characterized at the rat (or otherrelevant species analog) so that data generated from in vivo models canbe interpreted accurately. While the current panel of peptides revealedno major differences in pharmacological profile between the human Y2 andrat Y2a receptor analogs, even a single amino acid difference betweenreceptors displaying high sequence similarity could have dramaticeffects on ligand binding affinity (109). The rat Y2b receptorrepresents an additional opportunity to evaluate species-dependentdifferences in ligand binding. It remains to be determined whether therat Y2b receptor plays a singular role in rat Y2 receptor pharmacology,due either to unique ligand binding properties or to distinctivelocalization patterns.

[0230] We established functional assays for human Y2 receptor activationin both 293 and NIH-3T3 cells based on receptor-dependent inhibition offorskolin-stimulated cAMP accumulation (Table 9). The EC₅₀ values forpeptides in these assays were generally smaller than the correspondingK_(i) values, suggesting that receptor activation occurs through a highaffinity state of the receptor which is not predominantly representedunder the conditions of the binding assay. Such a scenario would beconsistent with the weak effect of Gpp(NH)p on radioligand binding tothe human Y2 receptor in membrane homogenates.

[0231] our characterization of the Y2 receptor stably expressed in 293cells also shows definitively that the Y2 receptor can couplesimultaneously to both cAMP regulation and calcium mobilization in asingle cell type. The calcium mobilization in 293 cells, at least,appears to occur through a pertussis toxin-sensitive G protein. The EC₅₀for the human PYY-mediated calcium response is significantly larger thanthat for the cAMP response in the same host cell (39 nM vs. 0.31 nM,respectively), suggesting that calcium mobilization requires promiscuouscoupling of the receptor to a G protein other than that involved incyclase regulation. The exact identities of the G proteins mediatingthese receptor activation events, whether G_(i), G_(o), G_(z), oranother type, remain to be determined.

[0232] We now have several Y2 receptor expression systems from which tochoose, each uniquely suited to different uses. The transient expressionsystem in COS-7, for example, allows us to generate sufficientquantities of membranes for routine structure/activity relationshipmeasurements. We can also produce mutant receptors by site-directedmutagenesis or related enzymatic techniques and express them transientlyin COS-7 for a comparison of pharmacological properties with those ofthe wild-type receptor. In this way, we can gain insight into receptorbinding pockets, ligand binding domains, and mechanisms of activation.The stable expression system in 293 and NIH-3T3 cells offers theconvenience of a single transfection followed by routine passagingtechniques. The stable expression system also offers the opportunity toselect for optimum receptor expression levels, G protein populations,and signal transduction pathways, all of which are critical elements forin vitro functional assays. Such assays can be used to determine agonistor antagonist activity in receptor-selective compounds, therebygenerating critical information for drug design.

[0233] The expression cloning of a human Y2 receptor allows, for thefirst time, the ability to develop NPY-receptor subtype specific drugsand represents a major advance in our ability to analyze NPY-mediatedphysiological processes. Pharmacologically defined Y2 receptors have awidespread anatomical distribution (2). They represent the predominantNPY receptor in brain, with the highest density in hippocampus andrelatively high expression in almost all other areas including olfactorybulb, basal ganglia, amygdaloid complex, thalamic and hypothalamicnuclei, pituitary, pineal gland, cerebellum, and brainstem. Thisdistribution is consistent with northern blot analysis, which shows thatthe Y2 mRNA is present in amygdala, candate nucleus, corpus callosum,hippocampus, hypothalamus, substantia nigra and subthalamic nucleus.Peripheral localization includes sympathetic neurons, dorsal rootganglia, stomach chief cells, intestinal enterocytes, kidney proximaltubule, trachea, and vascular smooth muscle. Y2 receptors are thereforein a position to potentially regulate a variety of physiologicalfunctions including cognitive enhancement, circadian rhythm, EEGsynchronization, body temperature, blood pressure, locomotor activity,neuroendocrine release, sympathetic activation, sensory transmission,gastrointestinal function, intestinal secretion, renal absorption, andcardiovascular function (1, 2).

[0234] Y2 receptors are attractive targets for drug design (1). Y2receptor regulation may be useful in the treatment of severalpathophysiological conditions (1, 2) including memory loss (111),epileptic seizure (72), pain (64), depression, hypertension, locomotorproblems, sleep disturbances, eating disorders, sexual/reproductivedisorders, nasal congestion (97), and diarrhea (112). A rigorousinvestigation of Y2-related pathophysiology has been hindered by theabsence of suitable non-peptide ligands. The chemical synthesis ofsubtype selective agonists and antagonists as potential drug candidateswill be greatly accelerated by screening against a homogeneouspopulation of cloned human Y2 receptors. As more specificpharmacological tools become available for probing receptor function,additional therapeutic indications are likely to be discovered.

[0235] We do not know whether the human and rat Y2 receptors we havediscovered account for all of the pharmacological Y2 receptors so fardescribed, or whether the Y2 receptor population is further divided intodistinct receptor subtypes. Indeed, there is some suggestion of receptorheterogeneity within the Y2 receptor population (2). These are issueswhich can now be resolved using nucleotide sequence from the human Y2receptor as the basis for in situ localization, anti-sense strategies,homology cloning, and related techniques. Such approaches will enable usto investigate the existence of potentially novel NPY receptor subtypes,in humans and other species, with additional pharmacologic andtherapeutic significance. TABLE 1 % aminoacid TM identity of the NPY-2receptor with other 7 TM Receptors m MUSGIR 42 h Y-1 41 h Y-4 41 h 5HT1A28 h Adenosine A2b 28 h Substance K 33 h 5HT2 31 h Adenosine A1 29 hSubstance P 32 h α-adrenergic-1b 34 h Dopamine D1 31 h Neurokinin-3 33 hα-adrenergic-2a 34 h Dopamine D2 32 h Interleukin-8 33 h β-adrenergic-135 bov Hist H1 25 h Angiotensin₁ 33 h Hist H2 28 h Angiotensin₂ 27 mThyrotropin releasing hor- 27 mone h Bradykinin 25 r mas oncogene 20

[0236] TABLE 2 Pharmacologically defined receptors for NPY and relatedpancreatic polypeptides. Affinity (−pK₁ or −pEC₅₀) Receptor 11 to 10 10to 9 9 to 8 8 to 7 7 to 6 <6 Y1 NPY NPY₂₋₃₆ NPY₁₃₋₃₆ PP PYY[Leu³¹,Pro³⁴]NPY Y2 PYY NPY₁₃₋₃₆ [Leu³¹,Pro³⁴]NPY NPY PP NPY₂₋₃₆ Y3 NPY[Pro³⁴]NPY NPY₁₃₋₃₆ PYY PP PP PP [Leu³¹,Pro³⁴]NPY NPY

[0237] TABLE 3 Pharmacological profile of the CG-13 receptor. Bindingdata reflect competitive displacement of ¹²⁵I-PYY from membranes ofCOS-7 cells transiently expressing CG-13 receptors. Pep- tides weretested at concentrations ranging from 0.001 nM to 100 nM. IC₅₀ valuescorresponding to 50% displacement were determined by nonlinearregression analysis and converted to K_(i) values acc- ording to theequation, K_(i) = IC₅₀/(1 + [L]/ K_(d)), where [L] is the ¹²⁵I-PYYconcentration and K_(d) is the equilibrium dissociation constant of¹²⁵I- PYY. The data shown are representative of at least two independentex- periments. SK-N- Human Y1, CG-13, Be(2), Competitor K_(i) (nM) K_(i)(nM) K_(i) (nM) human PYY 0.085 ± 0.021 0.39 ± 0.05 0.11 ± 0.02 humanNPY 0.049 ± 0.009 0.69 ± 0.14 0.13 ± 0.02 porcine 1.4 ± 0.2 0.78 ± 0.130.41 ± 0.09 NPY₂₋₃₆ porcine NPY 0.049 ± 0.001 0.86 ± 0.13 0.28 ± 0.04porcine 32 ± 7  1.5 ± 0.2 0.86 ± 0.14 PYY₁₃₋₃₆ porcine 28 ± 5  1.5 ± 0.22.1 ± 0.5 NPY₁₈₋₃₆ porcine 51 ± 16 2.4 ± 0.4 1.8 ± 0.4 NPY₁₃₋₃₆ porcine62 ± 6  3.4 ± 0.3 3.1 ± 0.6 NPY₂₀₋₃₆ porcine 45 ± 4  3.8 ± 0.7 5.0 ± 0.5NPY₁₆₋₃₆ porcine 170 ± 30  4.6 ± 0.1 3.2 ± 0.6 NPY₂₂₋₃₆ porcine >300 210± 60  70 ± 7  NPY₂₆₋₃₆ human NPY >300 >300 280 ± 120 free acid human PP200 ± 70  >300 >300 human 0.13 ± 0.02 >300 >300 [Leu³¹, Pro³⁴] NPY

[0238] TABLE 4 Extended pharmacological binding profile of the human Y2receptor vs. other Y-type receptors cloned from human. Binding datareflect competitive displacement of ¹²⁵I-PYY from membranes of COS-7cells transiently expressing human Y1, human Y2, and human Y4 receptors.IC₅₀ values corresponding to 50% displacement were determined bynonlinear regression analysis and converted to K_(i) values according tothe equation Chang-Prusoff equation, K_(i) = IC₅₀/(1 + [L]/K_(d)), where[L] is the ¹²⁵I-PYY concentration and K_(d) is the equilibriumdissociation constant of ¹²⁵I- PYY. Any peptide not included in initialcharacterization shown in previous tables is referred to as a “newpeptide”. Data shown are representative of at least two independentexperiments. Peptide Y1 Y2 Y4 Comments NPY, human 0.08 0.74 2.2 NPY,0.07 0.81 1.1 porcine NPY, frog 0.07 0.87 1.2 new (melanostatin) peptideO-Me-Tyr²¹- 0.12 1.6 6.1 new NPY, human peptide C2-NPY, 73 3.5 120 newporcine peptide NPY₂₋₃₆, 3.6 2.0 16 new human peptide NPY₂₋₃₆, 2.4 1.25.6 porcine NPY₁₃₋₃₆, 70 2.5 38 porcine NPY₁₆₋₃₆, 41 3.6 54 porcineNPY₁₈₋₃₆, 70 4.2 >300 porcine NPY₂₀₋₃₆, 63 3.6 120 porcineNPY₂₂₋₃₆, >1000 18 >990 porcine NPY₂₆₋₃₆, >1000 380 300 porcine [Leu³¹,0.15 >130 1.1 Pro³⁴]NPY, human [Leu³¹, 0.15 >540 1.5 new Pro³⁴]NPY,peptide procine NPY free 490 >1000 >1000 acid, humanNPY₁₋₂₄ >1000 >1000 >1000 new amide, peptide human [D- >1000 >1000 >1000new Trp³²]NPY, peptide human PYY, human 0.19 0.36 0.87 PYY, 0.14 0.351.3 new procine peptide PYY₃₋₃₆, 45 0.70 14 new human peptide PYY₁₃₋₃₆,33 1.5 46 porcine [Pro³⁴]PYY, 0.14 >310 0.12 new human peptide PP, human77 >1000 0.06 PP, bovine 240 >830 0.05 new peptide PP, rat 460 >10000.18 new peptide PP, avian 400 >1000 7.0 new peptide PP, frog 98 >100061 new peptide PP, salmon 0.20 0.17 3.2 new peptide [Ile³¹, >86 20 0.09new Gln^(34])PP, peptide human PYX-1 507 684 794 new peptidePYX-2 >1000 >1000 >1000 new peptide

[0239] TABLE 5 Macrolocalization of human Y2 receptor mRNA in humantissues by PCR. Localization data reflect PCR-based amplification ofhuman Y2 cDNA derived from mRNA extracts of human tissues. Southernblots of the PCR products were prepared and hybridized with ³²P-labeledoligonucleotide probes selective for Y-type receptor subtypes. Thelabaled products were recorded on X-ray film and the relative signaldensity was determined by visual inspection. In this rating scehem, + =faint signal, ++ = moderate signal +++ = intense signal. Human tissuesHuman Y2 PCR Product total brain +++ frontal brain ++ ventricle (heart)++ atrium (heart) (−) thoracic aorta +++ coronary artery ++½ nasalmucosa + mesentery ++ stomach ++ ileum ++ pancreas + liver (−) kidney +bladder +½ penis +++ testes not determined uterus (−) (endometrium)uterus (myometrium) (−)

[0240] TABLE 6 Peptide binding profile of the rat Y2a receptor vs. thehuman Y2 receptor. Binding data reflect competitive displacement of¹²⁵I-PYY from membranes of COS-7 cells transiently expressing rat Y2aand human Y2 receptors. IC₅₀ values corresponding to 50% displacementwere determined by nonlinear regression analysis and converted to K_(i)values according to the equation Chang-Prusoff equation, K_(i) =IC₅₀/(1 + [L]/K_(d)), where [L] is the ¹²⁵I-PYY concentration and K_(d)is the equilibrium dissociation constant of ¹²⁵I-PYY. Data shown arerepresentative of at least two independent experiments. Peptide Rat Y2aHuman Y2 NPY, human 1.3 0.74 NPY₂₋₃₆, human 2.2 1.2 NPY₁₃₋₃₆, human 312.5 NPY₂₀₋₃₆, porcine 93 3.6 NPY₂₆₋₃₆, porcine >830 380 NPY freeacid, >980 >1000 human [Leu³¹, Pro³⁴]NPY, >1000 >130 human[D-Trp³²]NPY, >830 >1000 human PYY, procine 0.28 0.35 PYY₁₃₋₃₆, procine1.5 28 PP, human >1000 >1000 PP₃₁₋₃₆, human >10,000 >10,000 PP, salmon0.17 0.17 PP, bovine >1000 >825 PP, rat >1000 >1000

[0241] TABLE 7 Oligonucleotide probe sequences used for in situhybridization Probe Sequence Location Orientation KS972 5′-GGC CCA TTAGGT GCA NH₂- sense GAG GCA GAT GAG AAT terminus CAA ACT GTA GAA GTG-3′KS974 5′-CAC TTC TAC AGT TTG NH₂- antisense ATT CTC ATC TGC CTC terminusTGC ACC TAA TGG GCC-3′ KS973 5′-CGG AGG TGT CCA TGA COOH sense CCT TCAAGG CTA AAA terminus AGA ACC TGC AAG TCA-3′ KS975 5′-TGA CTT CCA GGT TCTCOOH antisense TTT TAG CCT TGA AGG terminus TCA TGG ACA CCT CCG-3′

[0242] TABLE 8 Distribution of NPY Y2 mRNA in the rat CNS. Positivehybridization signals are indicated by “+” signs, no signal by “−”, anda low signal by “+/−”. Region Hybiridization Cortex layer 2 − layer 6 −piriform + enthorhinal − cingulate − Olfactory bulb − Anterior olfactoryn. − Basal ganglia caudate-putamen +/− n. accumbens − olfactorytubercle + glubus pallidus − islands of Calleja − Septal area lateralseptum + medial septum − septohippocampal − diagonal band n. − Claustrum− Dorsal endopiriform − Hypothalamus anterior − paraventricular +dorsomedial + ventromedial + arcuate + lateral − mammillary + tuberal +Thalamus anterior nuclei − paraventricular n. + rhomboid n. − reuniensn. − mediodorsal n. − ventral nuclei − reticular n. − cnetrolateral n. −centromedial n. + zona incerta − lateral posterior n. − lateral dorsaln. − posterior n. − medial geniculate n. − dorsal lateral gen. − ventrallateral gen. − habenula − Hippocampus CA1 − CA2 − CA3 + subiculum −presubiculum − parasubiculum − Dentate gyrus granule cell layer −polymorph layer − Amygdala anterior − medial + cortical + amygdalohipp.− basomedial + basolateral − lateral − central + bed nucleus − Midbrainsuperior colliculus − inferior colliculus − mes. trigeminal − dorsalraphe + caudal linear raphe + median raphe − raphe magnu − substantianigra − central gray − Pons/medulla locus coeruleus − subcoeruleus −parabrachial n. − facial n. − pontine n. + pontine ret. n. −reticulotegmental + A5 − A7 − gigantocellular − lateral reticular n. −motor trigeminal NA spinal trigeminal NA medial vestibular − solitariusNA dorsal vagus NA hypoglossal NA Cerebellum granule cell layer −molecular layer − Purkinje cells − deep nuclei − Spinal cord dorsal horn− ventral horn + intermediolateral − Dorsal root ganglia +

[0243] TABLE 9 Functional activation of the human Y2 receptor andinhibition of cAMP accumulation. K_(i) values were derived from bindingassays as described in Table 1. Peptides were evaluated for bindingaffinity and then analyzed for functional activity,. Functional datawere derived from radioimmunoassay of cAMP accumulation in stablytransfected 293 cells stimulated with 10 μM forskin. The maximuminhibition of cAMP accumulation relative to that produced by human NPY(E_(max)) and the concentration producing a half-maximal effect (EC₅₀)were determined by nonlinear regression. Data shown are representativeof at least two independent experiments. Function Binding EC₅₀ PeptideK_(i) (nM) (nM) E_(max) NPY, human 0.74 0.25 100% NPY, porcine 0.81 0.20113% C2-NPY, 3.5 0.14 116% porcine NPY₂₋₃₆, 2.0 0.35  94% human NPY₂₋₃₆,1.2 1.2  96% porcine NPY₁₃₋₃₆, 2.5 1.7 110% porcine NPY₁₆₋₃₆, 3.6 1.8 92% porcine NPY₁₈₋₃₆, 4.2 2.1  92% porcine NPY₂₀₋₃₆, 3.6 3.2  77%porcine NPY₂₂₋₃₆, 18 2.3  88% porcine [Leu⁺, >130 >3000 not Pro³⁴]NPY,determined human [Leu³¹, >540 >3000 not Pro³⁴]NPY, determined porcine[D-Trp³²]NPY, >1000 >3000 not human determined PYY, human 0.36 0.31 100%PYY, porcine 0.35 0.16 103% PYY₃₋₃₆, 0.70 0.22  99% human PYY₁₃₋₃₆, 1.50.13 102% porcine [Pro³⁴]PYY, >310 >120 not human determined PP, salmon0.17 0.07  79% PYX-1 684 >3000 not determined PYX-2 >1000 >3000 notdetermined

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1 27 1280 base pairs nucleic acid single linear cDNA CDS 43..1185 1GACTCTTGTG CTGGTTGCAG GCCAAGTGGA CCTGTACTGA AA ATG GGT CCA ATA 54 MetGly Pro Ile 1 GGT GCA GAG GCT GAT GAG AAC CAG ACA GTG GAA GAA ATG AAGGTG GAA 102 Gly Ala Glu Ala Asp Glu Asn Gln Thr Val Glu Glu Met Lys ValGlu 5 10 15 20 CAA TAC GGG CCA CAA ACA ACT CCT AGA GGT GAA CTG GTC CCTGAC CCT 150 Gln Tyr Gly Pro Gln Thr Thr Pro Arg Gly Glu Leu Val Pro AspPro 25 30 35 GAG CCA GAG CTT ATA GAT AGT ACC AAG CTG ATT GAG GTA CAA GTTGTT 198 Glu Pro Glu Leu Ile Asp Ser Thr Lys Leu Ile Glu Val Gln Val Val40 45 50 CTC ATA TTG GCC TAC TGC TCC ATC ATC TTG CTT GGG GTA ATT GGC AAC246 Leu Ile Leu Ala Tyr Cys Ser Ile Ile Leu Leu Gly Val Ile Gly Asn 5560 65 TCC TTG GTG ATC CAT GTG GTG ATC AAA TTC AAG AGC ATG CGC ACA GTA294 Ser Leu Val Ile His Val Val Ile Lys Phe Lys Ser Met Arg Thr Val 7075 80 ACC AAC TTT TTC ATT GCC AAT CTG GCT GTG GCA GAT CTT TTG GTG AAC342 Thr Asn Phe Phe Ile Ala Asn Leu Ala Val Ala Asp Leu Leu Val Asn 8590 95 100 ACT CTG TGT CTA CCG TTC ACT CTT ACC TAT ACC TTA ATG GGG GAGTGG 390 Thr Leu Cys Leu Pro Phe Thr Leu Thr Tyr Thr Leu Met Gly Glu Trp105 110 115 AAA ATG GGT CCT GTC CTG TGC CAC CTG GTG CCC TAT GCC CAG GGCCTG 438 Lys Met Gly Pro Val Leu Cys His Leu Val Pro Tyr Ala Gln Gly Leu120 125 130 GCA GTA CAA GTA TCC ACA ATC ACC TTG ACA GTA ATT GCC CTG GACCGG 486 Ala Val Gln Val Ser Thr Ile Thr Leu Thr Val Ile Ala Leu Asp Arg135 140 145 CAC AGG TGC ATC GTC TAC CAC CTA GAG AGC AAG ATC TCC AAG CGAATC 534 His Arg Cys Ile Val Tyr His Leu Glu Ser Lys Ile Ser Lys Arg Ile150 155 160 AGC TTC CTG ATT ATT GGC TTG GCC TGG GGC ATC AGT GCC CTG CTGGCA 582 Ser Phe Leu Ile Ile Gly Leu Ala Trp Gly Ile Ser Ala Leu Leu Ala165 170 175 180 AGT CCC CTG GCC ATC TTC CGG GAG TAT TCG CTG ATT GAG ATCATC CCG 630 Ser Pro Leu Ala Ile Phe Arg Glu Tyr Ser Leu Ile Glu Ile IlePro 185 190 195 GAC TTT GAG ATT GTG GCC TGT ACT GAA AAG TGG CCT GGC GAGGAG AAG 678 Asp Phe Glu Ile Val Ala Cys Thr Glu Lys Trp Pro Gly Glu GluLys 200 205 210 AGC ATC TAT GGC ACT GTC TAT AGT CTT TCT TCC TTG TTG ATCTTG TAT 726 Ser Ile Tyr Gly Thr Val Tyr Ser Leu Ser Ser Leu Leu Ile LeuTyr 215 220 225 GTT TTG CCT CTG GGC ATT ATA TCA TTT TCC TAC ACT CGC ATTTGG AGT 774 Val Leu Pro Leu Gly Ile Ile Ser Phe Ser Tyr Thr Arg Ile TrpSer 230 235 240 AAA TTG AAG AAC CAT GTC AGT CCT GGA GCT GCA AAT GAC CACTAC CAT 822 Lys Leu Lys Asn His Val Ser Pro Gly Ala Ala Asn Asp His TyrHis 245 250 255 260 CAG CGA AGG CAA AAA ACC ACC AAA ATG CTG GTG TGT GTGGTG GTG GTG 870 Gln Arg Arg Gln Lys Thr Thr Lys Met Leu Val Cys Val ValVal Val 265 270 275 TTT GCG GTC AGC TGG CTG CCT CTC CAT GCC TTC CAG CTTGCC GTT GAC 918 Phe Ala Val Ser Trp Leu Pro Leu His Ala Phe Gln Leu AlaVal Asp 280 285 290 ATT GAC AGC CAG GTC CTG GAC CTG AAG GAG TAC AAA CTCATC TTC ACA 966 Ile Asp Ser Gln Val Leu Asp Leu Lys Glu Tyr Lys Leu IlePhe Thr 295 300 305 GTG TTC CAC ATC ATC GCC ATG TGC TCC ACT TTT GCC AATCCC CTT CTC 1014 Val Phe His Ile Ile Ala Met Cys Ser Thr Phe Ala Asn ProLeu Leu 310 315 320 TAT GGC TGG ATG AAC AGC AAC TAC AGA AAG GCT TTC CTCTCG GCC TTC 1062 Tyr Gly Trp Met Asn Ser Asn Tyr Arg Lys Ala Phe Leu SerAla Phe 325 330 335 340 CGC TGT GAG CAG CGG TTG GAT GCC ATT CAC TCT GAGGTG TCC GTG ACA 1110 Arg Cys Glu Gln Arg Leu Asp Ala Ile His Ser Glu ValSer Val Thr 345 350 355 TTC AAG GCT AAA AAG AAC CTG GAG GTC AGA AAG AACAGT GGC CCC AAT 1158 Phe Lys Ala Lys Lys Asn Leu Glu Val Arg Lys Asn SerGly Pro Asn 360 365 370 GAC TCT TTC ACA GAG GCT ACC AAT GTC TAAGGAAGCTGTGGTGTGAA 1205 Asp Ser Phe Thr Glu Ala Thr Asn Val 375 380 AATGTATGGATGAATTCTGA CCAGAGCTAT GAATCTGGTT GATGGCGGCT CACAAGTGAA 1265 AACTGATTTCCCATT 1280 381 amino acids amino acid linear protein 2 Met Gly Pro IleGly Ala Glu Ala Asp Glu Asn Gln Thr Val Glu Glu 1 5 10 15 Met Lys ValGlu Gln Tyr Gly Pro Gln Thr Thr Pro Arg Gly Glu Leu 20 25 30 Val Pro AspPro Glu Pro Glu Leu Ile Asp Ser Thr Lys Leu Ile Glu 35 40 45 Val Gln ValVal Leu Ile Leu Ala Tyr Cys Ser Ile Ile Leu Leu Gly 50 55 60 Val Ile GlyAsn Ser Leu Val Ile His Val Val Ile Lys Phe Lys Ser 65 70 75 80 Met ArgThr Val Thr Asn Phe Phe Ile Ala Asn Leu Ala Val Ala Asp 85 90 95 Leu LeuVal Asn Thr Leu Cys Leu Pro Phe Thr Leu Thr Tyr Thr Leu 100 105 110 MetGly Glu Trp Lys Met Gly Pro Val Leu Cys His Leu Val Pro Tyr 115 120 125Ala Gln Gly Leu Ala Val Gln Val Ser Thr Ile Thr Leu Thr Val Ile 130 135140 Ala Leu Asp Arg His Arg Cys Ile Val Tyr His Leu Glu Ser Lys Ile 145150 155 160 Ser Lys Arg Ile Ser Phe Leu Ile Ile Gly Leu Ala Trp Gly IleSer 165 170 175 Ala Leu Leu Ala Ser Pro Leu Ala Ile Phe Arg Glu Tyr SerLeu Ile 180 185 190 Glu Ile Ile Pro Asp Phe Glu Ile Val Ala Cys Thr GluLys Trp Pro 195 200 205 Gly Glu Glu Lys Ser Ile Tyr Gly Thr Val Tyr SerLeu Ser Ser Leu 210 215 220 Leu Ile Leu Tyr Val Leu Pro Leu Gly Ile IleSer Phe Ser Tyr Thr 225 230 235 240 Arg Ile Trp Ser Lys Leu Lys Asn HisVal Ser Pro Gly Ala Ala Asn 245 250 255 Asp His Tyr His Gln Arg Arg GlnLys Thr Thr Lys Met Leu Val Cys 260 265 270 Val Val Val Val Phe Ala ValSer Trp Leu Pro Leu His Ala Phe Gln 275 280 285 Leu Ala Val Asp Ile AspSer Gln Val Leu Asp Leu Lys Glu Tyr Lys 290 295 300 Leu Ile Phe Thr ValPhe His Ile Ile Ala Met Cys Ser Thr Phe Ala 305 310 315 320 Asn Pro LeuLeu Tyr Gly Trp Met Asn Ser Asn Tyr Arg Lys Ala Phe 325 330 335 Leu SerAla Phe Arg Cys Glu Gln Arg Leu Asp Ala Ile His Ser Glu 340 345 350 ValSer Val Thr Phe Lys Ala Lys Lys Asn Leu Glu Val Arg Lys Asn 355 360 365Ser Gly Pro Asn Asp Ser Phe Thr Glu Ala Thr Asn Val 370 375 380 1556base pairs nucleic acid single linear DNA (genomic) NO NO CDS 211..13533 GTTGTTAACA GACTCGTGTA AAGGATTTGC TTTATGGAGC TTTTATGAGA TCTGTGGTGT 60GATGAATCAG AACACAGCTA CGCAGAGGAG CTCAGCCTAA ACTAAATCAA CCCCTTTAGG 120ATGGTTCTCT GTTTCACTAA CTTTTTTTAA TGTCGTTTTC TGTTATAGAT TCTTGTGCTA 180TCTGCAGGCC AAATTGGAAC TGAGGTGAAG ATG GGC CCA TTA GGT GCA GAG GCA 234 MetGly Pro Leu Gly Ala Glu Ala 1 5 GAT GAG AAT CAA ACT GTA GAA GTG AAA GTGGAA CTC TAT GGG TCG GGG 282 Asp Glu Asn Gln Thr Val Glu Val Lys Val GluLeu Tyr Gly Ser Gly 10 15 20 CCC ACC ACT CCT AGA GGT GAG TTG CCC CCT GATCCA GAG CCG GAG CTC 330 Pro Thr Thr Pro Arg Gly Glu Leu Pro Pro Asp ProGlu Pro Glu Leu 25 30 35 40 ATA GAC AGC ACC AAA CTG GTT GAG GTG CAG GTGGTC CTT ATA CTG GCC 378 Ile Asp Ser Thr Lys Leu Val Glu Val Gln Val ValLeu Ile Leu Ala 45 50 55 TAT TGT TCC ATC ATC TTG CTG GGC GTA GTT GGC AACTCT CTG GTA ATC 426 Tyr Cys Ser Ile Ile Leu Leu Gly Val Val Gly Asn SerLeu Val Ile 60 65 70 CAT GTG GTG ATC AAA TTC AAG AGC ATG CGC ACA GTA ACCAAC TTT TTT 474 His Val Val Ile Lys Phe Lys Ser Met Arg Thr Val Thr AsnPhe Phe 75 80 85 ATT GCC AAC CTG GCT GTG GCG GAT CTT TTG GTG AAC ACC CTGTGC CTG 522 Ile Ala Asn Leu Ala Val Ala Asp Leu Leu Val Asn Thr Leu CysLeu 90 95 100 CCA TTC ACT CTT ACC TAT ACC TTG ATG GGG GAG TGG AAA ATGGGT CCA 570 Pro Phe Thr Leu Thr Tyr Thr Leu Met Gly Glu Trp Lys Met GlyPro 105 110 115 120 GTT TTG TGC CAT TTG GTG CCC TAT GCC CAG GGT CTG GCAGTA CAA GTG 618 Val Leu Cys His Leu Val Pro Tyr Ala Gln Gly Leu Ala ValGln Val 125 130 135 TCC ACA ATA ACT TTG ACA GTC ATT GCT TTG GAC CGA CATCGT TGC ATT 666 Ser Thr Ile Thr Leu Thr Val Ile Ala Leu Asp Arg His ArgCys Ile 140 145 150 GTC TAC CAC CTG GAG AGC AAG ATC TCC AAG CAA ATC AGCTTC CTG ATT 714 Val Tyr His Leu Glu Ser Lys Ile Ser Lys Gln Ile Ser PheLeu Ile 155 160 165 ATT GGC CTG GCG TGG GGT GTC AGC GCT CTG CTG GCA AGTCCC CTT GCC 762 Ile Gly Leu Ala Trp Gly Val Ser Ala Leu Leu Ala Ser ProLeu Ala 170 175 180 ATC TTC CGG GAG TAC TCA CTG ATT GAG ATT ATT CCT GACTTT GAG ATT 810 Ile Phe Arg Glu Tyr Ser Leu Ile Glu Ile Ile Pro Asp PheGlu Ile 185 190 195 200 GTA GCC TGT ACT GAG AAA TGG CCC GGG GAG GAG AAGAGT GTG TAC GGT 858 Val Ala Cys Thr Glu Lys Trp Pro Gly Glu Glu Lys SerVal Tyr Gly 205 210 215 ACA GTC TAC AGC CTT TCC ACC CTG CTA ATC CTC TACGTT TTG CCT CTG 906 Thr Val Tyr Ser Leu Ser Thr Leu Leu Ile Leu Tyr ValLeu Pro Leu 220 225 230 GGC ATC ATA TCT TTC TCC TAC ACC CGG ATC TGG AGTAAG CTA AAG AAC 954 Gly Ile Ile Ser Phe Ser Tyr Thr Arg Ile Trp Ser LysLeu Lys Asn 235 240 245 CAC GTT AGT CCT GGA GCT GCA AGT GAC CAT TAC CATCAG CGA AGG CAC 1002 His Val Ser Pro Gly Ala Ala Ser Asp His Tyr His GlnArg Arg His 250 255 260 AAA ACG ACC AAA ATG CTC GTG TGC GTG GTA GTG GTGTTT GCA GTC AGC 1050 Lys Thr Thr Lys Met Leu Val Cys Val Val Val Val PheAla Val Ser 265 270 275 280 TGG CTG CCC CTC CAT GCC TTC CAA CTT GCT GTGGAC ATC GAC AGC CAT 1098 Trp Leu Pro Leu His Ala Phe Gln Leu Ala Val AspIle Asp Ser His 285 290 295 GTC CTG GAC CTG AAG GAG TAC AAA CTC ATC TTCACC GTG TTC CAC ATT 1146 Val Leu Asp Leu Lys Glu Tyr Lys Leu Ile Phe ThrVal Phe His Ile 300 305 310 ATT GCG ATG TGC TCC ACC TTC GCC AAC CCC CTTCTC TAT GGC TGG ATG 1194 Ile Ala Met Cys Ser Thr Phe Ala Asn Pro Leu LeuTyr Gly Trp Met 315 320 325 AAC AGC AAC TAC AGA AAA GCT TTC CTC TCA GCCTTC CGC TGT GAG CAG 1242 Asn Ser Asn Tyr Arg Lys Ala Phe Leu Ser Ala PheArg Cys Glu Gln 330 335 340 AGG TTG GAT GCC ATT CAC TCG GAG GTG TCC ATGACC TTC AAG GCT AAA 1290 Arg Leu Asp Ala Ile His Ser Glu Val Ser Met ThrPhe Lys Ala Lys 345 350 355 360 AAG AAC CTG GAA GTC AAA AAG AAC AAT GGCCTC ACT GAC TCT TTT TCA 1338 Lys Asn Leu Glu Val Lys Lys Asn Asn Gly LeuThr Asp Ser Phe Ser 365 370 375 GAG GCC ACC AAC GTG TAAGAATGCTGTGAAAGTAC GTGGGTAAAT TGCGACCAGA 1393 Glu Ala Thr Asn Val 380 GTTGCCAACCTGGTTAGGGA AGGTTTTCTG GCTAGTGCAT GCCACCTCCC ATTGTATTGA 1453 CCCTAAAAGCATCAGAGTGG AAGCCCCAGC GGTATTGTTC CTGGAAAACT GGCTGGAAGA 1513 ATGAGGAGAAAATAAACAGA TTGCTGTGGC GCAACGTTCT GAT 1556 381 amino acids amino acidlinear protein 4 Met Gly Pro Leu Gly Ala Glu Ala Asp Glu Asn Gln Thr ValGlu Val 1 5 10 15 Lys Val Glu Leu Tyr Gly Ser Gly Pro Thr Thr Pro ArgGly Glu Leu 20 25 30 Pro Pro Asp Pro Glu Pro Glu Leu Ile Asp Ser Thr LysLeu Val Glu 35 40 45 Val Gln Val Val Leu Ile Leu Ala Tyr Cys Ser Ile IleLeu Leu Gly 50 55 60 Val Val Gly Asn Ser Leu Val Ile His Val Val Ile LysPhe Lys Ser 65 70 75 80 Met Arg Thr Val Thr Asn Phe Phe Ile Ala Asn LeuAla Val Ala Asp 85 90 95 Leu Leu Val Asn Thr Leu Cys Leu Pro Phe Thr LeuThr Tyr Thr Leu 100 105 110 Met Gly Glu Trp Lys Met Gly Pro Val Leu CysHis Leu Val Pro Tyr 115 120 125 Ala Gln Gly Leu Ala Val Gln Val Ser ThrIle Thr Leu Thr Val Ile 130 135 140 Ala Leu Asp Arg His Arg Cys Ile ValTyr His Leu Glu Ser Lys Ile 145 150 155 160 Ser Lys Gln Ile Ser Phe LeuIle Ile Gly Leu Ala Trp Gly Val Ser 165 170 175 Ala Leu Leu Ala Ser ProLeu Ala Ile Phe Arg Glu Tyr Ser Leu Ile 180 185 190 Glu Ile Ile Pro AspPhe Glu Ile Val Ala Cys Thr Glu Lys Trp Pro 195 200 205 Gly Glu Glu LysSer Val Tyr Gly Thr Val Tyr Ser Leu Ser Thr Leu 210 215 220 Leu Ile LeuTyr Val Leu Pro Leu Gly Ile Ile Ser Phe Ser Tyr Thr 225 230 235 240 ArgIle Trp Ser Lys Leu Lys Asn His Val Ser Pro Gly Ala Ala Ser 245 250 255Asp His Tyr His Gln Arg Arg His Lys Thr Thr Lys Met Leu Val Cys 260 265270 Val Val Val Val Phe Ala Val Ser Trp Leu Pro Leu His Ala Phe Gln 275280 285 Leu Ala Val Asp Ile Asp Ser His Val Leu Asp Leu Lys Glu Tyr Lys290 295 300 Leu Ile Phe Thr Val Phe His Ile Ile Ala Met Cys Ser Thr PheAla 305 310 315 320 Asn Pro Leu Leu Tyr Gly Trp Met Asn Ser Asn Tyr ArgLys Ala Phe 325 330 335 Leu Ser Ala Phe Arg Cys Glu Gln Arg Leu Asp AlaIle His Ser Glu 340 345 350 Val Ser Met Thr Phe Lys Ala Lys Lys Asn LeuGlu Val Lys Lys Asn 355 360 365 Asn Gly Leu Thr Asp Ser Phe Ser Glu AlaThr Asn Val 370 375 380 1200 base pairs nucleic acid single lineargenomic DNA NO NO CDS 55..1200 5 TTTCTGTTAT AGATTCTTGT GCTATCTGCAGGCCAAATTG GAACTGAGGT GAAG ATG 57 Met 1 GGC CCA TTA GGT GCA GAG GCA GATGAG AAT CAA ACT GTA GAA GTG AAA 105 Gly Pro Leu Gly Ala Glu Ala Asp GluAsn Gln Thr Val Glu Val Lys 5 10 15 GTG GAA TTC TAT GGG TCG GGG CCC ACCACT CCT AGA GGT GAG TTG CCC 153 Val Glu Phe Tyr Gly Ser Gly Pro Thr ThrPro Arg Gly Glu Leu Pro 20 25 30 CCT GAT CCA GAG CCG GAG CTC ATA GAC AGCACC AAA CTG GTT GAG GTG 201 Pro Asp Pro Glu Pro Glu Leu Ile Asp Ser ThrLys Leu Val Glu Val 35 40 45 CAG GTG GTC CTT ATA CTG GCC TAT TGT TCC ATCATC TTG CTG GGC GTA 249 Gln Val Val Leu Ile Leu Ala Tyr Cys Ser Ile IleLeu Leu Gly Val 50 55 60 65 GTT GGC AAC TCT CTG GTA ATC CAT GTG GTG ATCAAA TTC AAG AGC ATG 297 Val Gly Asn Ser Leu Val Ile His Val Val Ile LysPhe Lys Ser Met 70 75 80 CGC ACA GTA ACC AAC TTT TTT ATT GCC AAC CTG GCTGTG GCG GAT CTT 345 Arg Thr Val Thr Asn Phe Phe Ile Ala Asn Leu Ala ValAla Asp Leu 85 90 95 TTG GTG AAC ACC CTG TGC CTG CCA TTC ACT CTT ACC TATACC TTG ATG 393 Leu Val Asn Thr Leu Cys Leu Pro Phe Thr Leu Thr Tyr ThrLeu Met 100 105 110 GGG GAG TGG AAA ATG GGT CCA GTT TTG TGC CAT TTG GTGCCC TAT GCC 441 Gly Glu Trp Lys Met Gly Pro Val Leu Cys His Leu Val ProTyr Ala 115 120 125 CAG GGT CTG GCA GTA CAA GTG TCC ACA ATA ACT TTG ACAGTC ATT GCT 489 Gln Gly Leu Ala Val Gln Val Ser Thr Ile Thr Leu Thr ValIle Ala 130 135 140 145 TTG GAC CGA CAT CGT TGC ATT GTC TAC CAC CTG GAGAGC AAG ATC TCC 537 Leu Asp Arg His Arg Cys Ile Val Tyr His Leu Glu SerLys Ile Ser 150 155 160 AAG CAA ATC AGC TTC CTG ATT ATT GGC CTG GCG TGGGGT GTC AGC GCT 585 Lys Gln Ile Ser Phe Leu Ile Ile Gly Leu Ala Trp GlyVal Ser Ala 165 170 175 CTG CTG GCA AGT CCC CTT GCC ATC TTC CGG GAG TACTCA CTG ATT GAG 633 Leu Leu Ala Ser Pro Leu Ala Ile Phe Arg Glu Tyr SerLeu Ile Glu 180 185 190 ATT ATT CCT GAC TTT GAG ATT GTA GCC TGT ACT GAGAAA TGG CCC GGG 681 Ile Ile Pro Asp Phe Glu Ile Val Ala Cys Thr Glu LysTrp Pro Gly 195 200 205 GAG GAG AAG AGT GTG TAC GGT ACA GTC TAC AGC CTTTCC ACC CTG CTA 729 Glu Glu Lys Ser Val Tyr Gly Thr Val Tyr Ser Leu SerThr Leu Leu 210 215 220 225 ATC CTC TAC GTT TTG CCT CTG GGC ATC ATA TCTTTC TCC TAC ACC CGG 777 Ile Leu Tyr Val Leu Pro Leu Gly Ile Ile Ser PheSer Tyr Thr Arg 230 235 240 ATC TGG AGT AAG CTA AAG AAC CAC GTT AGT CCTGGA GCT GCA AGT GAC 825 Ile Trp Ser Lys Leu Lys Asn His Val Ser Pro GlyAla Ala Ser Asp 245 250 255 CAT TAC CAT CAG CGA AGG CAC AAA ATG ACC AAAATG CTC GTG TGC GTG 873 His Tyr His Gln Arg Arg His Lys Met Thr Lys MetLeu Val Cys Val 260 265 270 GTA GTG GTG TTT GCA GTC AGC TGG CTG CCC CTCCAT GCC TTC CAA CTT 921 Val Val Val Phe Ala Val Ser Trp Leu Pro Leu HisAla Phe Gln Leu 275 280 285 GCT GTG GAC ATC GAC AGC CAT GTC CTG GAC CTGAAG GAG TAC AAA CTC 969 Ala Val Asp Ile Asp Ser His Val Leu Asp Leu LysGlu Tyr Lys Leu 290 295 300 305 ATC TTC ACC GTG TTC CAC ATT ATT GCG ATGTGC TCC ACC TTC GCC AAC 1017 Ile Phe Thr Val Phe His Ile Ile Ala Met CysSer Thr Phe Ala Asn 310 315 320 CCC CTT CTC TAT GGC TGG ATG AAC AGC AACTAC AGA AAA GCT TTC CTC 1065 Pro Leu Leu Tyr Gly Trp Met Asn Ser Asn TyrArg Lys Ala Phe Leu 325 330 335 TCA GCC TTC CGC TGT GAG CAG AGG TTG GATGCC ATT CAC TCG GAG GTG 1113 Ser Ala Phe Arg Cys Glu Gln Arg Leu Asp AlaIle His Ser Glu Val 340 345 350 TCC ATG ACC TTC AAG GCT AAA AAG AAC CTGGAA GTC AAA AAG AAC AAT 1161 Ser Met Thr Phe Lys Ala Lys Lys Asn Leu GluVal Lys Lys Asn Asn 355 360 365 GGC CTC ACT GAC TCT TTT TCA GAG GCC ACCAAC GTG TAA 1200 Gly Leu Thr Asp Ser Phe Ser Glu Ala Thr Asn Val * 370375 380 381 amino acids amino acid linear protein 6 Met Gly Pro Leu GlyAla Glu Ala Asp Glu Asn Gln Thr Val Glu Val 1 5 10 15 Lys Val Glu PheTyr Gly Ser Gly Pro Thr Thr Pro Arg Gly Glu Leu 20 25 30 Pro Pro Asp ProGlu Pro Glu Leu Ile Asp Ser Thr Lys Leu Val Glu 35 40 45 Val Gln Val ValLeu Ile Leu Ala Tyr Cys Ser Ile Ile Leu Leu Gly 50 55 60 Val Val Gly AsnSer Leu Val Ile His Val Val Ile Lys Phe Lys Ser 65 70 75 80 Met Arg ThrVal Thr Asn Phe Phe Ile Ala Asn Leu Ala Val Ala Asp 85 90 95 Leu Leu ValAsn Thr Leu Cys Leu Pro Phe Thr Leu Thr Tyr Thr Leu 100 105 110 Met GlyGlu Trp Lys Met Gly Pro Val Leu Cys His Leu Val Pro Tyr 115 120 125 AlaGln Gly Leu Ala Val Gln Val Ser Thr Ile Thr Leu Thr Val Ile 130 135 140Ala Leu Asp Arg His Arg Cys Ile Val Tyr His Leu Glu Ser Lys Ile 145 150155 160 Ser Lys Gln Ile Ser Phe Leu Ile Ile Gly Leu Ala Trp Gly Val Ser165 170 175 Ala Leu Leu Ala Ser Pro Leu Ala Ile Phe Arg Glu Tyr Ser LeuIle 180 185 190 Glu Ile Ile Pro Asp Phe Glu Ile Val Ala Cys Thr Glu LysTrp Pro 195 200 205 Gly Glu Glu Lys Ser Val Tyr Gly Thr Val Tyr Ser LeuSer Thr Leu 210 215 220 Leu Ile Leu Tyr Val Leu Pro Leu Gly Ile Ile SerPhe Ser Tyr Thr 225 230 235 240 Arg Ile Trp Ser Lys Leu Lys Asn His ValSer Pro Gly Ala Ala Ser 245 250 255 Asp His Tyr His Gln Arg Arg His LysMet Thr Lys Met Leu Val Cys 260 265 270 Val Val Val Val Phe Ala Val SerTrp Leu Pro Leu His Ala Phe Gln 275 280 285 Leu Ala Val Asp Ile Asp SerHis Val Leu Asp Leu Lys Glu Tyr Lys 290 295 300 Leu Ile Phe Thr Val PheHis Ile Ile Ala Met Cys Ser Thr Phe Ala 305 310 315 320 Asn Pro Leu LeuTyr Gly Trp Met Asn Ser Asn Tyr Arg Lys Ala Phe 325 330 335 Leu Ser AlaPhe Arg Cys Glu Gln Arg Leu Asp Ala Ile His Ser Glu 340 345 350 Val SerMet Thr Phe Lys Ala Lys Lys Asn Leu Glu Val Lys Lys Asn 355 360 365 AsnGly Leu Thr Asp Ser Phe Ser Glu Ala Thr Asn Val 370 375 380 49 aminoacids nucleic acid single linear DNA (genomic) NO NO 7 CAAGTTGTTCTCATATTGGC CTACTGCTCC ATCATCTTGC TTGGGGTAAT 50 49 amino acids nucleicacid single linear DNA (genomic) NO NO 8 ATCACCACAT GGATCACCAAGGAGTTGCCA ATTACCCCAA GCAAGATGAT 50 44 amino acids nucleic acid singlelinear DNA (genomic) NO NO 9 TTTTTCATTG CCAATCTGGC TGTGGCAGAT CTTTTGGTGAACACT 45 44 amino acids nucleic acid single linear DNA (genomic) NO NO10 AGGTAAGAGT GAACGGTAGA CACAGAGTGT TCACCAAAAG ATCTG 45 44 amino acidsnucleic acid single linear DNA (genomic) NO NO 11 CCACCTGGTG CCCTATGCCCAGGGCCTGGC AGTACAAGTA TCCAC 45 44 amino acids nucleic acid single linearDNA (genomic) NO NO 12 CAGGGCAATT ACTGTCAAGG TGATTGTGGA TACTTGTACT GCCAG45 44 amino acids nucleic acid single linear DNA (genomic) NO NO 13AATCAGCTTC CTGATTATTG GCTTGGCCTG GGGCATCAGT GCCCT 45 44 amino acidsnucleic acid single linear DNA (genomic) NO NO 14 GAAGATGGCC AGGGGACTTGCCAGCAGGGC ACTGATGCCC CAGGC 45 44 amino acids nucleic acid single linearDNA (genomic) NO NO 15 ACTGTCTATA GTCTTTCTTC CTTGTTGATC TTGTATGTTT TGCCT45 44 amino acids nucleic acid single linear DNA (genomic) NO NO 16TGTAGGAAAA TGATATAATG CCCAGAGGCA AAACATACAA GATCA 45 44 amino acidsnucleic acid single linear DNA (genomic) NO NO 17 CTGGTGTGTG TGGTGGTGGTGTTTGCGGTC AGCTGGCTGC CTCTC 45 44 amino acids nucleic acid single linearDNA (genomic) NO NO 18 TGTCAACGGC AAGCTGGAAG GCATGGAGAG GCAGCCAGCT GACCG45 46 amino acids nucleic acid single linear DNA (genomic) NO NO 19CTCATCTTCA CAGTGTTCCA CATCATCGCC ATGTGCTCCA CTTTTGC 47 46 amino acidsnucleic acid single linear DNA (genomic) NO NO 20 TTCATCCAGC CATAGAGAAGGGGATTGGCA AAAGTGGAGC ACATGGC 47 24 amino acids nucleic acid singlelinear DNA (genomic) NO NO 21 GGGAGTATTC GCTGATTGAG ATCAT 25 22 aminoacids nucleic acid single linear DNA (genomic) NO NO 22 GCCTTGAATGTCACGGACAC CTC 23 44 amino acids nucleic acid single linear DNA(genomic) NO NO 23 CTGATGGTAG TGGTCATTTG CAGCTCCAGG ACTGACATGG TTCTT 4544 amino acids nucleic acid single linear DNA (genomic) NO 24 GGCCCATTAGGTGCAGAGGC AGATGAGAAT CAAACTGTAG AAGTG 45 44 amino acids nucleic acidsingle linear DNA (genomic) YES 25 CACTTCTACA GTTTGATTCT CATCTGCCTCTGCACCTAAT GGGCC 45 44 amino acids nucleic acid single linear DNA(genomic) NO 26 CGGAGGTGTC CATGACCTTC AAGGCTAAAA AGAACCTGGA AGTCA 45 44amino acids nucleic acid single linear DNA (genomic) YES 27 TGACTTCCAGGTTCTTTTTA GCCTTGAAGG TCATGGACAC CTCCG 45

What is claimed is:
 1. An isolated nucleic acid molecule encoding a Y2receptor.
 2. An isolated nucleic acid molecule of claim 1, wherein thenucleic acid molecule is a DNA molecule.
 3. An isolated DNA molecule ofclaim 2, wherein the DNA molecule is a cDNA molecule.
 4. An isolated DNAmolecule of claim 2, wherein the DNA molecule is a genomic DNA molecule.5. An isolated nucleic acid molecule of claim 1, wherein the nucleicacid molecule is a RNA molecule.
 6. An isolated nucleic acid molecule ofclaim 1 wherein the nucleic acid molecule encodes a human Y2 receptor.7. An isolated nucleic acid molecule of claim 6 wherein the nucleic acidmolecule encodes a receptor being characterized by an amino acidsequence in the transmembrane region, which amino acid sequence has 60%homology or higher to the amino acid sequence in the transmembraneregion of the human Y2 receptor shown in FIG.
 11. 8. An isolated nucleicacid molecule of claim 6 wherein the human Y2 receptor has substantiallythe same amino acid sequence as shown in FIG.
 2. 9. An isolated nucleicacid molecule of claim 6 wherein the human Y2 receptor has the aminoacid sequence as shown in FIG.
 2. 10. An isolated nucleic acid moleculeof claim 1 wherein the nucleic acid molecule encodes a rat Y2 receptor.11. An isolated nucleic acid molecule of claim 10 wherein the rat Y2receptor has substantially the same amino acid sequence as shown in FIG.8.
 12. An isolated nucleic acid molecule of claim 10 wherein the rat Y2receptor has the amino acid sequence shown in FIG.
 8. 13. An isolatednucleic acid molecule of claim 10 wherein the rat Y2 receptor hassubstantially the same amino acid sequence as shown in FIG.
 9. 14. Anisolated nucleic acid molecule of claim 10 wherein the rat Y2 receptorhas the amino acid sequence shown in FIG.
 9. 15. An isolated, purifiedY2 receptor protein.
 16. A vector comprising the nucleic acid moleculeof claim
 1. 17. A vector comprising the nucleic acid molecule of claim6.
 18. A vector comprising the nucleic acid molecule of claim
 10. 19. Avector of claim 16 adapted for expression in a bacterial cell whichcomprises the regulatory elements necessary for expression of thenucleic acid in the bacterial cell operatively linked to the nucleicacid encoding the Y2 receptor as to permit expression thereof.
 20. Avector of claim 16 adapted for expression in a yeast cell whichcomprises the regulatory elements necessary for expression of thenucleic acid in the yeast cell operatively linked to the nucleic acidencoding the Y2 receptor as to permit expression thereof.
 21. A vectorof claim 16 adapted for expression in an insect cell which comprises theregulatory elements necessary for expression of the nucleic acid in theinsect cell operatively linked to the nucleic acid encoding the Y2receptor as to permit expression thereof.
 22. A vector of claim 21wherein the vector is a baculovirus.
 23. A vector of claim 16 adaptedfor expression in a mammalian cell which comprises the regulatoryelements necessary for expression of the nucleic acid in the mammaliancell operatively linked to the nucleic acid encoding the Y2 receptor asto permit expression thereof.
 24. A vector of claim 17 adapted forexpression in a mammalian cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the mammalian celloperatively linked to the nucleic acid encoding the Y2 receptor as topermit expression thereof.
 25. A vector of claim 24 wherein the vectoris a plasmid.
 26. The plasmid of claim 25 designated pcEXV-hY2 (ATCCAccession No. 75659).
 27. A vector of claim 18 adapted for expression ina mammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the Y2 receptor as to permit expressionthereof.
 28. A vector of claim 27 wherein the vector is a plasmid. 29.The plasmid of claim 28 designated pcEXV-rY2a (ATCC Accession No.97035).
 30. The plasmid of claim 28 designated pcEXV-rY2b (ATCCAccession No. 97036).
 31. A cell comprising the vector of either ofclaims 24 or
 28. 32. The cell of claim 31 wherein the cell is amammalian cell.
 33. The cell of claim 32 wherein the mammalian cell isnon-neuronal in origin.
 34. The cell of claim 33 wherein the mammaliancell non-neuronal in origin is a COS-7 cell.
 35. The cell of claim 33wherein the mammalian cell non-neuronal in origin is a NIH-3T3 cell. 36.A NIH-3T3 cell of claim 36 designated N-hY2-5 (ATCC Accession No.CRL-11825).
 37. The cell of claim 33 wherein the mammalian cellnon-neuronal in origin is a 293 human embryonic kidney cell.
 38. A 293human embryonic kidney cell of claim 37 designated 293-hY2-10 (ATCCAccession No. 11837).
 39. A nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a unique sequence included within the sequence of a nucleic acidmolecule encoding a Y2 receptor.
 40. The nucleic acid probe of claim 39wherein the nucleic acid is DNA.
 41. The nucleic acid probe of claim 39wherein the nucleic acid encodes a human Y2 receptor.
 42. The nucleicacid probe of claim 39 wherein the nucleic acid encodes a rat Y2receptor.
 43. An antisense oligonucleotide having a sequence capable ofspecifically hybridizing to an mRNA molecule encoding a Y2 receptor soas to prevent translation of the mRNA molecule.
 44. An antisenseoligonucleotide having a sequence capable of specifically hybridizing tothe cDNA molecule of claim
 3. 45. An antisense oligonucleotide of eitherof claims 43 or 44 comprising chemical analogues of nucleotides.
 46. Anantibody directed to a Y2 receptor.
 47. An antibody of claim 46, whereinthe Y2 receptor is a human Y2 receptor.
 48. An antibody of claim 46wherein the Y2 receptor is a rat Y2 receptor.
 49. An antibody of claim46, wherein the antibody is a monoclonal antibody.
 50. A monoclonalantibody of claim 49 directed to an epitope of a Y2 receptor present onthe surface of a Y2 receptor expressing cell.
 51. A pharmaceuticalcomposition comprising an amount of the oligonucleotide of claim 43effective to decrease activity of a Y2 receptor by passing through acell membrane and binding specifically with mRNA encoding a Y2 receptorin the cell so as to prevent its translation and a pharmaceuticallyacceptable carrier capable of passing through a cell membrane.
 52. Apharmaceutical composition of claim 51, wherein the oligonucleotide iscoupled to a substance which inactivates mRNA.
 53. A pharmaceuticalcomposition of claim 52, wherein the substance which inactivates mRNA isa ribozyme.
 54. A pharmaceutical composition of claim 51, wherein thepharmaceutically acceptable carrier comprises a structure which binds toa receptor on a cell capable of being taken up by cells after binding tothe structure.
 55. A pharmaceutical composition of claim 54, wherein thestructure of the pharmaceutically acceptable carrier is capable ofbinding to a receptor which is specific for a selected cell type.
 56. Apharmaceutical composition comprising an amount of the antibody of claim46 effective to block binding of a ligand to a Y2 receptor and apharmaceutically acceptable carrier.
 57. A transgenic nonhuman mammalexpressing nucleic acid encoding a Y2 receptor.
 58. A transgenicnonhuman mammal comprising a homologous recombination knockout of thenative Y2 receptor.
 59. A transgenic nonhuman mammal whose genomecomprises antisense nucleic acid complementary to nucleic acid encodinga Y2 receptor so placed as to be transcribed into antisense mRNA whichis complementary to mRNA encoding a Y2 receptor and which hybridizes tomRNA encoding a Y2 receptor thereby reducing its translation.
 60. Thetransgenic nonhuman mammal of either of claims 57 or 59, wherein thenucleic acid encoding a Y2 receptor additionally comprises an induciblepromoter.
 61. The transgenic nonhuman mammal of either of claims 57 or59, wherein the nucleic acid encoding a Y2 receptor additionallycomprises tissue specific regulatory elements.
 62. A transgenic nonhumanmammal of any of claims 57, 58 or 59, wherein the transgenic nonhumanmammal is a mouse.
 63. A method for determining whether a ligand canbind specifically to a Y2 receptor which comprises contacting a celltransfected with and expressing nucleic acid encoding the Y2 receptorwith the ligand under conditions permitting binding of ligands to suchreceptor, and detecting the presence of any such ligand boundspecifically to the Y2 receptor, thereby determining whether the ligandbinds specifically to a Y2 receptor.
 64. A method of claim 63 whereinthe Y2 receptor is a human Y2 receptor.
 65. A method of claim 63 whereinthe Y2 receptor is a rat Y2 receptor.
 66. A method for determiningwhether a ligand can bind specifically to a Y2 receptor, which comprisescontacting a cell transfected with and expressing nucleic acid encodingthe Y2 receptor with the ligand under conditions permitting binding ofligands to such receptor, and detecting the presence of any such ligandspecifically bound to the Y2 receptor, thereby determining whether theligand binds specifically to a Y2 receptor, wherein the Y2 receptor ischaracterized by an amino acid sequence in the transmembrane region,such amino acid sequence having 60% homology or higher to the amino acidsequence in the transmembrane region of the Y2 receptor shown in FIG.11.
 67. A method of claim 66 wherein the Y2 receptor is a human Y2receptor.
 68. A method of claim 66 wherein the Y2 receptor is a rat Y2receptor.
 69. A method for determining whether a ligand can bindspecifically to a Y2 receptor which comprises preparing a cell extractfrom cells transfected with and expressing nucleic acid encoding the Y2receptor, isolating a membrane fraction from the cell extract,contacting the ligand with the membrane fraction under conditionspermitting binding of ligands to such receptor, and detecting thepresence of any ligand bound to the Y2 receptor, thereby determiningwhether the compound is capable of specifically binding to a Y2receptor.
 70. A method of claim 69 wherein the Y2 receptor is a human Y2receptor.
 71. A method of claim 69 wherein the Y2 receptor is a rat Y2receptor.
 72. A method of any of claims 63, 64, 65, 66, 67, 68, 69, 70,or 71 wherein the ligand is not previously known.
 73. A liganddetermined by the method of claim
 72. 74. A method for determiningwhether a ligand is a Y2 receptor agonist which comprises contacting acell transfected with and expressing nucleic acid encoding the Y2receptor with the ligand under conditions permitting the activation of afunctional Y2 receptor response from the cell, and detecting by means ofa bioassay, such as a second messenger assay, an increase in Y2 receptoractivity, thereby determining whether the ligand is a Y2 receptoragonist.
 75. A method for determining whether a ligand is a Y2 receptoragonist which comprises preparing a cell extract from cells transfectedwith and expressing nucleic acid encoding the Y2 receptor, isolating amembrane fraction from the cell extract, contacting the membranefraction of the extract with the ligand under conditions permitting theactivation of a functional Y2 receptor response, and detecting by meansof a bioassay, such as a second messenger assay, an increase in Y2receptor activity, thereby determining whether the ligand is a Y2receptor agonist.
 76. A method of either of claims 74 or 75 wherein theY2 receptor is a human Y2 receptor.
 77. A method of either of claims 74or 75 wherein the Y2 receptor is a rat Y2 receptor.
 78. A method fordetermining whether a ligand is a Y2 receptor antagonist which comprisescontacting a cell transfected with and expressing nucleic acid encodinga Y2 receptor with the ligand in the presence of a known Y2 receptoragonist, such as NPY, under. conditions permitting the activation of afunctional Y2 receptor response, and detecting by means of a bioassay,such as a second messenger assay, a decrease in Y2 receptor activity,thereby determining whether the ligand is a Y2 receptor antagonist. 79.A method for determining whether a ligand is a Y2 receptor antagonistwhich comprises preparing a cell extract from cells transfected with andexpressing nucleic acid encoding the Y2 receptor, isolating a membranefraction from the cell extract, contacting the membrane fraction of theextract with the ligand in the presence of a known Y2 receptor agonist,such as NPY, under conditions permitting the activation of a functionalY2 receptor response, and detecting by means of a bioassay, such as asecond messenger assay, a decrease in Y2 receptor activity, therebydetermining whether the ligand is a Y2 receptor antagonist.
 80. A methodof either of claims 78 or 79 wherein the Y2 receptor is a human Y2receptor.
 81. A method of either of claims 78 or 79 wherein the Y2receptor is a rat Y2 receptor.
 82. A method of any of claims 74, 75, 78,or 79 wherein the second messenger assay comprises measurement ofintracellular cAMP.
 83. A method of any of claims 74, 75, 78, or 79wherein the second messenger assay comprises measurement ofintracellular calcium mobilization.
 84. A method of any of claims 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or81 wherein the cell is a mammalian cell.
 85. A method of claim 84wherein the mammalian cell is nonneuronal in origin.
 86. A method ofclaim 85, wherein the mammalian cell is nonneuronal in origin is a COS-7cell.
 87. A method of claim 85, wherein the mammalian cell nonneuronalin origin is a 293 human embryonic kidney cell.
 88. The cell of claim 87designated 293-hY2-10 (ATCC Accession No. 11837).
 89. A method of claim85, wherein the mammalian cell nonneuronal in origin is a LM(tk-) cell.90. A method of claim 85, wherein the mammalian cell nonneuronal inorigin is a NIH-3T3 cell.
 91. A cell of claim 90 designated N-hY2-5(ATCC Accession No. CRL-11825).
 92. A ligand detected by the method ofany of claims 74, 75, 76, 77, 78, 79, 80, or
 81. 93. A ligand of claim92 wherein the ligand is not previously known.
 94. A pharmaceuticalcomposition comprising an amount of a Y2 receptor agonist determined bythe method of either of claims 74 or 75 effective to activate a Y2receptor and a pharmaceutically acceptable carrier.
 95. A pharmaceuticalcomposition of claim 94 wherein the Y2 receptor agonist is notpreviously known.
 96. A pharmaceutical composition which comprises anamount of a Y2 receptor antagonist determined by the method of either ofclaims 78 or 79 effective to decrease activity of a Y2 receptor and apharmaceutically acceptable carrier.
 97. A pharmaceutical composition ofclaim 96 wherein the Y2 receptor antagonist is not previously known. 98.A method of screening drugs to identify drugs which specifically bind toa Y2 receptor on the surface of a cell which comprises contacting a celltransfected with and expressing nucleic acid encoding the Y2 receptorwith a plurality of drugs under conditions permitting binding of drugsto the Y2 receptor;, and determining those drugs which bind specificallyto the transfected cell, thereby identifying drugs which bindspecifically to a Y2 receptor.
 99. A method of screening drugs toidentify drugs which bind specifically to a Y2 receptor on the surfaceof a cell which comprises preparing a cell extract from cellstransfected with and expressing nucleic acid encoding the Y2 receptor,isolating a membrane fraction from the cell extract, contacting themembrane fraction with a plurality of drugs under conditions permittingbinding of drugs to the Y2 receptor, and determining those drugs whichbind specifically to the transfected cell, thereby identifying drugswhich bind specifically to a Y2 receptor.
 100. A method of either ofclaims 98 or 99 wherein the Y2 receptor is a human Y2 receptor.
 101. Amethod of either of claims 98 or 99 wherein the Y2 receptor is a rat Y2receptor.
 102. A method of screening drugs to identify drugs which actas agonists of a Y2 receptor which comprises contacting a celltransfected with and expressing nucleic acid encoding the Y2 receptorwith a plurality of drugs under conditions permitting the activation ofa functional Y2 receptor response, and determining those drugs whichactivate such receptor using a bioassay, such as a second messengerassay, thereby identifying drugs which act as agonists of a Y2 receptor.103. A method of screening drugs to identify drugs which act as agonistsof a Y2 receptor which comprises preparing a cell extract from cellstransfected with and expressing nucleic acid encoding the Y2 receptor,isolating a membrane fraction from the cell extract, contacting themembrane fraction with a plurality of drugs under conditions permittingthe activation of a functional Y2 receptor response, and determiningthose drugs which activate such receptor using a bioassay, such as asecond messenger assay, thereby identifying drugs which act as agonistsof a Y2 receptor.
 104. A method of either of claims 102 or 103 whereinthe Y2 receptor is a human Y2 receptor.
 105. A method of either ofclaims 102 or 103 wherein the Y2 receptor is a rat Y2 receptor.
 106. Amethod of screening drugs to identify drugs which act as antagonists ofY2 receptors which comprises contacting a cell transfected with andexpressing nucleic acid encoding a Y2 receptor with a plurality of drugsin the presence of a known Y2 receptor agonist such as NPY underconditions permitting the activation of a functional Y2 receptorresponse, and determining those drugs which inhibit the activation ofthe receptor using a bioassay, such as a second messenger assay, therebyidentifying drugs which act as antagonists of Y2 receptors.
 107. Amethod of screening drugs to identify drugs which act as antagonists ofY2 receptors which comprises preparing a cell extract from cellstransfected with and expressing nucleic acid encoding the Y2 receptor,isolating a membrane fraction from the cell extract, contacting themembrane fraction with a plurality of drugs in the presence of a knownY2 receptor agonist such as NPY under conditions permitting theactivation of a functional Y2 receptor response, and determining thosedrugs which inhibit the activation of the receptor using a bioassay,such as a second messenger assay, thereby identifying drugs which act asantagonists of Y2 receptors.
 108. A method of either of claims 106 or107 wherein the Y2 receptor is a human Y2 receptor.
 109. A method ofeither of claims 106 or 107 wherein the Y2 receptor is a rat Y2receptor.
 110. A method of any of claims 102, 103, 106 or 107 whereinthe second messenger assay comprises measurement of intracellular cAMP.111. A method of any of claims 102, 103, 106, or 107 wherein the secondmessenger assay comprises measurement of intracellular calciummobilization.
 112. A method of any of claims 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, or 109 wherein the cell is a mammalian cell.113. A method of claim 112 wherein the mammalian cell is nonneuronal inorigin.
 114. The method of claim 113 wherein the mammalian cellnonneuronal in origin is a Cos-7 cell.
 115. The method of claim 113wherein the mammalian cell nonneuronal in origin is a 293 humanembryonic kidney cell.
 116. The cell of claim 115 designated 293-hY2-10(ATCC Accession No. 11837).
 117. The method of claim 113 wherein themammalian cell nonneuronal in origin is a LM(tk-) cell.
 118. The methodof claim 113 wherein the mammalian cell nonneuronal in origin is aNIH-3T3 cell.
 119. The cell of claim 118 designated N-hY2-5 (ATCCAccession No. CRL-11825).
 120. A pharmaceutical composition comprisingan effective amount of a drug identified by the method of either ofclaims 102 or 103 and a pharmaceutically acceptable carrier.
 121. Apharmaceutical composition comprising an effective amount of a drugidentified by the method of either of claims 106 or 107 and apharmaceutically acceptable carrier.
 122. A method of detectingexpression of a Y2 receptor by a cell by detecting the presence of mRNAcoding for a Y2 receptor which comprises obtaining total mRNA from thecell and contacting the mRNA so obtained with the nucleic acid probe ofclaim 39 under hybridizing conditions, and detecting the presence ofmRNA hybridized to the probe, thereby detecting the expression of Y2receptor by the cell.
 123. A method of treating an abnormality in asubject, wherein the abnormality is alleviated by activation of a Y2receptor which comprises administering to a subject an effective amountof the pharmaceutical composition of either of claims 94 or 120, therebytreating the abnormality.
 124. A method of treating an abnormality in asubject, wherein the abnormality is alleviated by activation of a Y2receptor which comprises administering to a subject an effective amountof Y2 receptor agonist determined by any of claims 74, 75, 102, or 103,thereby treating the abnormality.
 125. A method of treating anabnormality in a subject, wherein the abnormality is alleviated bydecreasing the activity of a Y2 receptor which comprises administeringto a subject an effective amount of the pharmaceutical composition ofeither of claims 96 or 121, thereby treating the abnormality.
 126. Amethod of treating an abnormality in a subject, wherein the abnormalityis alleviated by decreasing the activity of a Y2 receptor whichcomprises administering to the subject an effective amount of a Y2receptor antagonist determined by the methods of any of claims 78, 79,106, or 107, thereby treating the abnormality.
 127. The method of eitherof claims 125 or 126 wherein the abnormality is a cognitive disorder.128. The method of either of claims 125 or 126 wherein the abnormalityis a gastrointestinal disorder.
 129. The method of either of claims 125or 126 wherein the abnormality is sleeping disorder.
 130. The method ofeither of claims 125 or 126 wherein the abnormality is epilepsy. 131.The method of either claims 125 or 126 wherein the abnormality ishypertension.
 132. The method of either of claims 123 or 124 wherein theabnormality is memory loss.
 133. The method of either of claims 123 or124 wherein the abnormality is diarrhea.
 134. The method of either ofclaims 123 or 124 wherein the abnormality is nasal congestion.
 135. Themethod of either of claims 123 or 124 wherein the abnormality is pain.136. A method of treating an abnormality in a subject, wherein theabnormality alleviated by decreasing the activity of a Y2 receptor whichcomprises administering to the subject an amount of the pharmaceuticalcomposition of claim 56 effective to block binding of ligands to the Y2receptor, thereby treating the abnormality.
 137. A method of treating anabnormality in a subject, wherein the abnormality is alleviated bydecreasing the activity of a Y2 receptor which comprises administeringto the subject an effective amount of the pharmaceutical composition ofclaim 51, thereby treating the abnormality.
 138. The method of either ofclaims 136 or 137 wherein the abnormality is a cognitive disorder. 139.The method of either of claims 136 or 137 wherein the abnormality is agastrointestinal disorder.
 140. The method of either of claims 136 or137 wherein the abnormality is epilepsy.
 141. The method of either ofclaims 136 or 137 wherein the abnormality is hypertension.
 142. Themethod of either of claims 136 or 137 wherein the abnormality issleeping disorder.
 143. A method of detecting the presence of a Y2receptor on the surface of a cell which comprises contacting the cellwith the antibody of claim 46 under conditions permitting binding of theantibody to the receptor, and detecting the presence of the antibodybound to the cell, thereby detecting the presence of a Y2 receptor onthe surface of the cell.
 144. A method of determining the physiologicaleffects of expressing varying levels of Y2 receptors which comprisesproducing a transgenic nonhuman mammal of claim 55 whose levels of humanY2 receptor expression are varied by use of an inducible promoter whichregulates Y2 receptor expression.
 145. A method of determining thephysiological effects of expressing varying levels of Y2 receptors whichcomprises producing a panel of transgenic nonhuman mammals of claim 55each expressing a different amount of Y2 receptor.
 146. A method foridentifying a Y2 receptor antagonist capable of alleviating anabnormality in a subject, wherein the abnormality is alleviated bydecreasing the activity of a Y2 receptor which comprises administeringthe antagonist to a transgenic nonhuman mammal of any of claims 57, 58,or 59 and determining whether the antagonist alleviates the physical andbehavioral abnormalities displayed by the transgenic nonhuman mammal asa result of activity of a Y2 receptor, thereby identifying a Y2antagonist.
 147. An antagonist identified by the method of claim 146.148. A pharmaceutical composition comprising an effective amount of anantagonist identified by the method of claim 146 and a pharmaceuticallyacceptable carrier.
 149. A method for treating an abnormality in asubject wherein the abnormality is alleviated by decreasing the activityof a Y2 receptor which comprises administering to the subject aneffective amount of the pharmaceutical composition of claim 148, therebytreating the abnormality.
 150. A method for identifying a Y2 receptoragonist capable of alleviating an abnormality wherein the abnormality isalleviated by activation of a Y2 receptor which comprises administeringthe agonist to the transgenic nonhuman mammal of any of claims 57, 58,or 59 and determining whether the agonist alleviates the physical andbehavioral abnormalities displayed by the transgenic nonhuman mammal,thereby identifying a Y4 receptor agonist.
 151. An agonist identified bythe method of claim
 150. 152. A pharmaceutical composition comprising aneffective amount of an agonist identified by the method of claim 150 anda pharmaceutically acceptable carrier.
 153. A method for treating anabnormality in a subject wherein the abnormality is alleviated byactivation of a Y2 receptor which comprises administering to the subjectan effective amount of the pharmaceutical composition of claim 152,thereby treating the abnormality.
 154. A method for diagnosing apredisposition to a disorder associated with the activity of a specificY2 receptor allele which comprises: a. obtaining nucleic acid ofsubjects suffering from the disorder; b. performing a restriction digestof the nucleic acid with a panel of restriction enzymes; c.electrophoretically separating the resulting nucleic acid fragments on asizing gel; d. contacting the resulting gel with a nucleic acid probecapable of specifically hybridizing to nucleic acid encoding a Y2receptor and labelled with a detectable marker; e. detecting labelledbands which have hybridized to the nucleic acid encoding a Y2 receptorlabelled with a detectable marker to create a unique band patternspecific to the nucleic acid of subjects suffering from the disorder; f.preparing nucleic acid obtained for diagnosis by steps a-e; and g.comparing the unique band pattern specific to the nucleic acid ofsubjects suffering from the disorder from step e and the nucleic acidobtained for diagnosis from step f to determine whether the patterns arethe same or different and to diagnose thereby predisposition to thedisorder if the patterns are the same.
 155. The method of claim 154wherein a disorder associated with the expression of a specific Y2receptor allele is diagnosed.
 156. A method of preparing the isolated,purified Y2 receptor of claim 15 which comprises: a. constructing avector adapted for expression in a cell which comprises the regulatoryelements necessary for the expression of nucleic acid in the celloperatively linked to the nucleic acid encoding a Y2 receptor as topermit expression thereof, wherein the cell is selected from the groupconsisting of bacterial cells, yeast cells, insect cells and mammaliancells; b. inserting the vector of step (a) in a suitable host cell; c.incubating the cells of step (b) under conditions allowing theexpression of a Y2′receptor; d. recovering the receptor so produced; e.purifying the receptor so recovered.